Synthetic modified rna and uses thereof

ABSTRACT

The present application relates to a nucleic acid molecule comprising a first nucleic acid sequence comprising at least a portion of a 5′ untranslated region (5′ UTR) of a carboxylesterase gene and a second nucleic acid sequence encoding a protein of interest, where the second nucleic acid sequence is heterologous to and operatively coupled to the first nucleic acid sequence. Also disclosed are methods of expressing a protein of interest in a target cell, methods of treating subject for cardiac ischemia or hepatic ischemia, and methods of identifying a nucleic acid sequence capable of selectively enhancing translation of a heterologous protein of interest in a target cell.

This application claims the priority benefit of U.S. Provisional PatentApplication Ser. No. 62/932,255, filed Nov. 7, 2019, which is herebyincorporated by reference in its entirety.

This invention was made with government support under RO1 HL142768-01awarded by National Institutes of Health. The government has certainrights in the invention.

FIELD

The present application relates to nucleic acid molecules andpharmaceutical compositions thereof, to methods involving the nucleicacid molecules and pharmaceutical compositions, and to methods ofidentifying a nucleic acid sequence capable of selectively enhancingtranslation of a heterologous protein of interest in a target cell.

BACKGROUND

Ischemic heart disease is the leading cause of death for both men andwomen in the U.S., killing about 610,000 people each year. Thus, it isdesirable to devise novel treatments that improve cardiac functionpost-ischemic injury. One avenue of research into treating a failingheart post-myocardial infarction (“MI”) is genetic medicine (Tilemann etal., “Gene Therapy for Heart Failure,” Circ. Res. 110:777-793 (2012)),by which scientists aim to adjust gene expression in the heart usingviral vectors, small molecules, or RNA-based approaches to promotecardiac protection as well as cardiovascular or cardiac regeneration inischemic cardiac disease. Synthetic modified messenger RNA (“modRNA”) isa novel gene therapy platform that can be used to alter protein levelsin mammalian cells and tissues (Sultana et al., “Optimizing CardiacDelivery of Modified mRNA,” Mol. Ther. 25(6):1306-1315 (2017) and Hadaset al., “Optimizing Modified mRNA In Vitro Synthesis Protocol for HeartGene Therapy,” Mol. Ther. Methods Clin. Dev. 14(13):300-305 (2019)) andto treat cardiac disease (Zangi et al., “Modified mRNA Directs the Fateof Heart Progenitor Cells and Induces Vascular Regeneration afterMyocardial Infarction,” Nat. Biotechnol. 31:898-907 (2013); Magadum &Zangi, “mRNA-Based Protein Replacement Therapy for the Heart,” Mol.Ther. 27:785-793 (20189); and Hadas et al., “Modified mRNA as aTherapeutic Tool to Induce Cardiac Regeneration in Ischemic HeartDisease,” Wiley Interdiscip. Rev. Syst. Biol. Med. 9(1):e1367 (2017)).The concept of therapeutically altering mRNA expression has greatpotential to treat human diseases (Weissman & Kariko, “mRNA: Fulfillingthe Promise of Gene Therapy,” Mol. Ther. 23:1416-1417 (2015)). To date,several therapeutic approaches using siRNA and antisenseoligonucleotides have been shown to reduce mRNA levels in cells (Bobbin& Rossi, “RNA Interference (RNAi)-Based Therapeutics: Delivering on thePromise?,” Annu. Rev. Pharmacol. Toxicol. 56:103-122 (2016) and Stein &Castanotto, “FDA-Approved Oligonucleotide Therapies in 2017,” Mol. Ther.25(5):1069-1075 (2017)). Yet, protein upregulation in tissues ischallenging, mostly due to the high amount of mRNA needed to treat humantissue. Supplying a large amount of mRNA in vivo can elicit undesirableimmune responses to the administered mRNA. Preclinical studies havesuggested that due to the transient expression of modRNA (target geneexpression returns to baseline within 48-72 hours of administration),both directly and intravenously delivered modRNA will need to beadministered multiple times to achieve desired levels of target geneexpression (Zangi et al., “Modified mRNA Directs the Fate of HeartProgenitor Cells and Induces Vascular Regeneration after MyocardialInfarction,” Nat. Biotechnol. 31:898-907 (2013); Pardi et al.,“Expression Kinetics of Nucleoside-Modified mRNA Delivered in LipidNanoparticles to Mice by Various Routes,” J. Control Release 217:345-351(2015); Mahiny et al., “In Vivo Genome Editing Using Nuclease-EncodingmRNA Corrects SP-B Deficiency,” Nat. Biotechnol. 33:584-586 (2015);Kormann et al., “Expression of Therapeutic Proteins after Delivery ofChemically Modified mRNA in Mice,” Nat. Biotechnol. 29:154-157 (2011);and Zimmermann et al., “Successful Use of mRNA-Nucleofection forOverexpression of Interleukin-10 in Murine Monocytes/Macrophages forAnti-Inflammatory Therapy in a Murine Model of Autoimmune Myocarditis,”J. Am. Heart Assoc. 1:e003293 (2012)). Thus, one obstacle in usingmodRNA for the treatment of cardiac ischemic disease is achieving highlevels of target protein expression by direct administration of modRNAto the heart. It is also desirable to achieve high levels of targetprotein expression following a single administration of modRNA.

Gene expression is controlled intricately at the post-transcriptionallevel (Mignone et al., “Untranslated Regions of mRNAs,” Genome Biol.3(3):REVIEWS0004.1 (2002)). The level of any individual mRNA type insidea cell does not ensure that comparable amounts of respective proteinswill be expressed (Vogel et al., “Sequence Signatures and mRNAConcentration Can Explain Two-Thirds of Protein Abundance Variation in aHuman Cell Line,” Mol. Syst. Biol. 6:400 (2010)). Both positive andnegative modulators influence translation and maintain certain proteinlevels. Within the untranslated regions (UTRs) of the mRNA are multipleregulatory elements, which are critical for mRNA stability andtranslation into protein (Pfeiffer et al., “Using TranslationalEnhancers to Increase Transgene Expression in Drosophila,” Proc. Natl.Acad. Sci. USA 109:6626-6631 (2012) and Wilkie et al., “Regulation ofmRNA Translation by 5′- and 3′-UTR-Binding Factors,” Trends Biochem.Sci. 28(4):182-188 (2003)). Eukaryotic gene translation is regulated atthe translation level by several components, including the 5′untranslated region (“5′ UTR”) (Ong et al., “The Role of 5′ UntranslatedRegion in Translational Suppression of OKL38 mRNA in HepatocellularCarcinoma,” Oncogene 26(8):1155-65 (2007); Leppek et al., “Functional 5′UTR mRNA Structures in Eukaryotic Translation Regulation and How to FindThem,” Nat. Rev. Mol. Cell Biol. 19(3):158-174 (2018); and van derVelden & Thomas, “The Role of the 5′ Untranslated Region of an mRNA inTranslation Regulation During Development,” Int. J. Biochem. Cell Biol.31(1):87-106 (1999)), the 3′ untranslated region (“3′ UTR”) (van Oers etal., “Role of the 3′ Untranslated Region of Baculovirus p10 mRNA inHigh-Level Expression of Foreign Genes,” J. Gen. Virol. 80(Pt8):2253-2262 (1999); Thekkumkara et al., “Functional Role for theAngiotensin II Receptor (AT1A) 3′-Untranslated Region in DeterminingCellular Responses to Agonist: Evidence for Recognition by RNA BindingProteins,” Biochem. J. 329(Pt 2):255-264 (1998); and Chen et al., “TheFunctional Role of the 3′ Untranslated Region and Poly(A) Tail of DuckHepatitis A Virus Type 1 in Viral Replication and Regulation ofIRES-Mediated Translation,” Front. Microbiol. 9:2250 (2018)), poly Atail (Chartier et al., “Mitochondrial Dysfunction Reveals the Role ofmRNA Poly(A) Tail Regulation in Oculopharyngeal Muscular DystrophyPathogenesis,” PLoS Genet. 11(3):e1005092 (2015); Crawford et al., “TheRole of 3′ Poly(A) Tail Metabolism in Tumor Necrosis Factor-AlphaRegulation,” J. Biol. Chem. 272(34):21120-21127 (1997); Nie et al.,“Sarcoplasmic Reticulum Ca2+ pump mRNA Stability in Cardiac and SmoothMuscle: Role of Poly A+ Tail Length,” Cell Calcium 35(5):479-84 (2004);and Peng et al., “Characterization of the Role of Hexamer AGUAAA andPoly(A) Tail in Coronavirus Polyadenylation,” PLoS One 11(10):e0165077(2016)), and cap structure (Galloway & Cowling, “mRNA Cap Regulation inMammalian Cell Function and Fate,” Biochim. Biophys. Acta Gene Regul.Mech. 1862(3):270-279 (2019); Grudzien-Nogalska et al., “Synthesis ofAnti-Reverse Cap Analogs (ARCAs) and Their Applications in mRNATranslation and Stability,” Methods Enzymol. 431:203-227 (2007); Meaux &Van Hoof, “Yeast Transcripts Cleaved by an Internal Ribozyme Provide NewInsight Into the Role of the Cap and Poly(A) Tail in Translation andmRNA Decay,” RNA 12(7):1323-1337 (2006); and Mukherj ee et al.,“Identification of Cytoplasmic Capping Targets Reveals a Role for CapHomeostasis in Translation and mRNA Stability,” Cell Rep. 2(3):674-684(2012)).

Moreover, the length and secondary structure of the 5′ UTR, as well asany mutations it contains, have been reported to be associated withcertain human diseases (Chatterjee & Pal, “Role of 5′- and3′-Untranslated Regions of mRNAs in Human Diseases,” Biol. Cell101(5):251-262 (2009)). The 5′ UTR plays a significant role inregulating translation efficiency by helping the ribosome bind themessenger RNA (“mRNA”) proximal to the start codon and thus is a maincontributor to the cellular proteome (Hinnebusch et al., “TranslationalControl by 5′-Untranslated Regions of Eukaryotic mRNAs,” Science352(6292):1413-1416 (2016)). Additionally, certain RNA elements withinthe 5′ UTR may change its secondary structure (e.g., internal ribosomeentry sites (IRES), upstream AUGs, or open reading frames (uORFs)) andcan be important contributors to the entire translation rate (Araujo etal., “Before It Gets Started: Regulating Translation at the 5′ UTR,”Comp. Funct. Genomics 2012:475731 (2012) and Dvir et al., “Decipheringthe Rules by which 5′-UTR Sequences Affect Protein Expression in Yeast,”Proc. Natl. Acad. Sci. USA 110(30):E2792-2801 (2013)). 5′ UTRs can alsocontain sequence elements that can function as binding sites forregulatory proteins (Wilkie et al., “Regulation of mRNA Translation by5′- and 3′-UTR-Binding Factors,” Trends Biochem. Sci. 28(4):182-188(2003)).

To date, the modRNA used in pre-clinical cardiac research has employedan artificial 5′ UTR (36 nucleotides) that was first described by Warrenet al., “Highly Efficient Reprogramming to Pluripotency and DirectedDifferentiation of Human Cells with Synthetic Modified mRNA,” Cell StemCell 7(5):618-630 (2010). In vitro screening has been used to optimizemRNA 5′ UTR and improve Arginase 1 (ARG1) expression (Asrani et al.,“Optimization of mRNA Untranslated Regions for Improved Expression ofTherapeutic mRNA,” RNA Biol. 15(6):756-762 (2018)). However,plasmid-based screening methods do not necessarily correlate withprotein expression driven by exogenously expressed mRNA (Asrani et al.,“Optimization of mRNA Untranslated Regions for Improved Expression ofTherapeutic mRNA,” RNA Biol. 15(6):756-762 (2018)). Moreover, improved5′ UTR, but not 3′ UTR, appears to be the key driver in proteinexpression for exogenously delivered mRNA (Asrani et al., “Optimizationof mRNA Untranslated Regions for Improved Expression of TherapeuticmRNA,” RNA Biol. 15(6):756-762 (2018)).

To this end, no screening approaches have been carried out to identifyalternative 5′ UTRs to improve mRNA translatability of in vitrosynthesized modRNA constructs of therapeutic interest.

The present application is directed to overcoming these and otherdeficiencies in the art.

SUMMARY

One aspect of the present application relates to a nucleic acid moleculecomprising a first nucleic acid sequence comprising at least a portionof a 5′ untranslated region (5′ UTR) of a carboxylesterase gene and asecond nucleic acid sequence encoding a protein of interest, where thesecond nucleic acid sequence is heterologous to and operatively coupledto the first nucleic acid sequence.

Another aspect of the present application relates to a pharmaceuticalcomposition comprising the nucleic acid molecule described herein.

A further aspect of the present application relates to a method ofexpressing a protein of interest in a target cell. This method involvesproviding a nucleic acid molecule or the pharmaceutical compositiondescribed herein and contacting a target cell with the nucleic acidmolecule or pharmaceutical composition, where the nucleic acid moleculeis translated to express the protein of interest in the target cell.

Another aspect of the present application relates to a method oftreating a subject for cardiac ischemia or hepatic ischemia. This methodinvolves providing the nucleic acid molecule or the pharmaceuticalcomposition described herein and contacting the subject with the nucleicacid molecule or the pharmaceutical composition described herein, wherethe nucleic acid molecule is translated to express a protein of interestin the subject's heart or liver to treat the subject for cardiacischemia or hepatic ischemia.

A further aspect of the present application relates to a method ofidentifying a 5′ untranslated region (5′ UTR) for selectively enhancingtranslation of a heterologous protein of interest in a target cell ortissue. This method involves obtaining a first sample of living tissuecomprising a target cell under disease conditions and a second sample ofliving tissue comprising a target cell under non-disease conditions;quantifying genes that are transcribed and translated in the first andsecond samples; identifying a gene which is (i) transcribed at similaror lower levels in the first sample relative to the second sample and(ii) translated at higher levels in the first sample relative to thesecond sample; and identifying the 5′ UTR of the identified gene, wherethe identified 5′ UTR is capable of selectively enhancing translation ofa heterologous protein of interest in a target cell or tissue.

Modified mRNA (modRNA) is a gene delivery platform for transientlyintroducing a single or several genes of interest to different celltypes and tissues. modRNA is considered a safe vector for gene transferas it negligibly activates the innate immune system and does notcompromise genome integrity. Due to its clinical potential, modRNA usein basic and translational science is rising. It is desirable to usemodRNA to induce cardiac regeneration post-ischemic injury. However,major obstacles in using modRNA for cardiac ischemic disease include theneed to directly, singly administer modRNA to the heart and theinefficient translation of modRNA due to its short half-life. Modulating5′ untranslated regions (5′ UTR) to enhance translation efficiency inischemic cardiac disease can reduce the amount of modRNA needed perdelivery by achieving higher and longer protein production post singledelivery. Described herein is the identification of the 5′ UTR from thefatty acid metabolism gene carboxylesterase 1D as capable of increasingmodRNA-mediated translation in the heart post-myocardial infarction. Theresults presented herein specifically identify the Ces1d RNA element(Element D) as responsible for the enhanced modRNA translationpost-ischemic injury in the heart. Importantly, the 5′ UTR of Ces1d wasfound to enhance modRNA translation in the heart and liver, but not inthe kidney, post-ischemic injury. These results suggest that the 5′ UTRof Ces1d and Element D of Ces1d play a wider role in protein translationunder ischemic conditions in different organs. These results also formthe foundation for the nucleic acid molecules and pharmaceuticalcompositions described herein, methods involving the nucleic acidmolecules and pharmaceutical compositions disclosed herein, and themethods of identifying a nucleic acid sequence capable of selectivelyenhancing translation of a heterologous protein of interest in a targetcell described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G demonstrate the characterization of the ischemic hearttranscriptome and proteome. FIG. 1A is a schematic diagram illustratingthe experimental protocol used to prepare samples for RNAseq andproteomic analysis. Sham-operated or LAD-ligated hearts were collected4-hours or 24-hours post-myocardial infarction (MI) and the ischemicarea tissue (or equivalent area in sham-operated hearts) was dividedinto two equal pieces. One-half of the ischemic heart was sequenced fortranscriptomic analysis (n=10 total, Sham n=3, 4-hours post-MI n=3,24-hours post-MI n=4), while the other half of the ischemic heart wasevaluated for protein level using mass spectrometry (n=12 total, Shamn=4, 4-hours post-MI n=4, 24-hours post-MI n=4). FIGS. 1B-1C arehierarchical clustering dendrograms of 2,272 genes with correspondingmRNA level (FIG. 1B) or 2,397 protein intensities (FIG. 1C) in Sham,4-hours post-MI, or 24-hours post-MI hearts. FIGS. 1D-1E are graphsshowing correlation analysis between changes in levels of proteins andmRNA in the LV 4-hours (FIG. 1D) or 24-hours (FIG. 1E) post-MI. Thebottom right shaded rectangles in FIG. 1D and FIG. 1E include genes thatshow static or reduced mRNA levels post-MI while their encoded proteinslevel increased in comparison to Sham. FIGS. 1F-1G are tables showing alist of genes that encode for proteins with elevated protein levels(fold change>2), but lowered mRNA (fold change<0.64) 4-hours (FIG. 1F)or 24-hours (FIG. 1G) post-MI. Genes in the light shaded boxes have a 5′UTR that is shorter than 100 base pairs.

FIGS. 2A-2D show that RNAseq experimental groups are clustered togetherin a hierarchical clustering dendrogram and Ces1d western blot confirmsRNAseq and proteomic expression results. FIG. 2A is a hierarchicalclustering dendrogram of 14,000 genes that were sequenced fortranscriptomic analysis from ischemic or non-ischemic heart tissues(n=10 total, Sham n=3, 4 hours post MI n=3 or 24 hours post MU n=4).FIG. 2B is a graph showing the results of a qPCR experiment used toevaluate Ces1d expression performed on samples taken 24 hours fromhearts that have undergone Sham or MU surgery (n-3). FIG. 2C is arepresentative image of western blot analysis performed with anti-Ces1dantibody to evaluate protein samples taken 24 hours from hearts thathave undergone Sham or MI surgery. FIG. 2D is a graph showing thequantification of the experiment shown in FIG. 2C (n=2). Unpairedtwo-tailed t-test for FIG. 2B and FIG. 2D. *, P<0.05.

FIGS. 3A-3F show the translation efficiency of various syntheticmodified mRNA (modRNA) constructs comprising 5′ UTRs from genesidentified in FIGS. 2A-2D. FIG. 3A is a schematic illustration of themodRNA constructs evaluated in FIGS. 3B-3F. The illustration shows thereplacement of a commonly used artificial 5′ UTR (i.e., a control 5′UTR) with the 5′ UTR of Gsn, Pzp, Serpina 1b, Fnk3, or Ces1d in a LucmodRNA reporter construct. FIG. 3B is a schematic diagram illustratingthe experimental plan to evaluate the translation efficiency of LucmodRNA or GFP modRNA comprising the 5′ UTRs from FIG. 3A in neonatal ratcardiomyocytes (CMs) using Interactive IVIS® or western blot analysis,respectively. FIGS. 3C-3D show the quantification of the IVIS® (FIG. 3C,n=4) and western blot (FIG. 3D) results described in FIG. 3B. FIG. 3E isa schematic diagram illustrating the experimental plan to use IVIS®analysis to evaluate the translation efficiency of Luc modRNA constructscomprising the 5′ UTRs from FIG. 3A in mouse hearts 24-hours post-MIusing IVIS® analysis. FIG. 3F is a graph showing the quantification ofthe IVIS® experiment described in FIG. 3E (n=4). One-way ANOVA, Tukey'sMultiple Comparison Test for FIG. 3C and FIG. 3F. ***, P<0.001, *,P<0.05, N.S., Not Significant.

FIGS. 4A-4E demonstrate in vitro IVIS® analysis of Luc modRNA inneonatal rat CMs. FIG. 4A is a schematic diagram illustrating theexperimental protocol for evaluating translation efficiency in neonatalrat CMs using Luc modRNA constructs (Luc-Control, Luc-Pzp, Luc-Gsn,Luc-Fn3k, Luc-Serpina 1b, Luc-Ces1d, and Renilla-Control) and IVIS®analysis. FIGS. 4B-4C show representative IVIS® analysis images of theluciferase (Luc) signal (FIG. 4B) and Renilla signal (FIG. 4C) from theexperiment described in FIG. 4A. FIGS. 4D-4E are graphs showing thequantification of the experimental results shown in FIGS. 4B-4C,respectively.

FIGS. 5A-5D demonstrate that the 5′ UTR of Ces1d significantly enhancesmRNA translation in ischemic hearts but not in a non-ischemic mousemodel. FIG. 5A is a schematic diagram illustrating the experimentalprotocol used to evaluate the translation efficiency of Luc modRNAcomprising the 5′ UTR of Ces1d (Luc-Ces1d) or an artificial control 5′UTR (Luc-Control) in a non-ischemic heart model. FIG. 5B is a graphquantifying the Luc signal observed 24 hours, 48 hours, and 72 hoursfollowing injection with Luc-Ces1d modRNA and Luc-Control modRNAfollowing the protocol of FIG. 5A (n=15). FIG. 5C is a schematic diagramillustrating the experimental protocol used to evaluate the translationefficiency of Luc modRNA comprising the 5′ UTR of Ces1d (Luc-Ces1d) oran artificial control 5′ UTR (Luc-Control) in an ischemic heart model.FIG. 5D is a graph showing quantification of the Luc signal observed 24hours, 48 hours, and 72 hours following LED ligation and modRNAinjection with Luc-Ces1d modRNA and Luc-Control modRNA following theprotocol of FIG. 5C (n=15). Two-way ANOVA, Tukey's Multiple ComparisonTest for FIG. 5B & FIG. 5D. *, P<0.05, N.S., Not Significant.

FIGS. 6A-6F demonstrate the translation efficiency of Luc-modRNAconstructs comprising the 5′ UTR of Ces1d (Luc-Ces1d) and an artificialcontrol 5′ UTR (Luc-Control) in ischemic heart, kidney, and liver mousemodels. FIG. 6A is a schematic diagram illustrating the experimentalprotocol used to evaluate the translation efficiency of Luc modRNAcomprising the 5′ UTR of Ces1d (Luc-Ces1d) or an artificial control 5′UTR (Luc-Control) in an ischemic heart mouse model. FIG. 6B showsrepresentative IVIS® imaging results of the experiment described in FIG.6A. FIG. 6C is a schematic diagram illustrating the experimentalprotocol used to evaluate the translation efficiency of Luc modRNAcomprising the 5′ UTR of Ces1d (Luc-Ces1d) or an artificial control 5′UTR (Luc-Control) in an ischemic liver mouse model. FIG. 6D shows arepresentative IVIS® image from the experiment described in FIG. 6C.FIG. 6E is a schematic diagram illustrating the experimental protocolused to evaluate the translation efficiency of Luc modRNA comprising the5′ UTR of Ces1d (Luc-Ces1d) or an artificial control 5′ UTR(Luc-Control) in an ischemic kidney mouse model. FIG. 6F shows arepresentative IVIS® image of the experiment described in FIG. 6E.

FIGS. 7A-7H demonstrate that the 5′ UTR of Ces1d significantly enhancesLuc modRNA translation in the liver, but not in non-ischemic or kidneyischemic mouse models. FIG. 7A is a schematic diagram illustrating theexperimental protocol used to evaluate the translation efficiency of LucmodRNA comprising the 5′ UTR of Ces1d (Luc-Ces1d) or an artificialcontrol 5′ UTR (Luc-Control) in a non-ischemic liver model. FIG. 7B is agraph showing quantification of the experiment described in FIG. 7A(n=6). FIG. 7C is a schematic diagram illustrating the experimentalprotocol used to evaluate the translation efficiency of Luc modRNAcomprising the 5′ UTR of Ces1d (Luc-Ces1d) or an artificial control 5′UTR (Luc-Control) in an ischemic liver model. FIG. 7D is a graph showingquantification of the experiment described in FIG. 7C (n=6). FIG. 7E isa schematic diagram illustrating the experimental plan to evaluate thetranslation efficiency of Luc modRNA comprising the 5′ UTR of Ces1d(Luc-Ces1d) or an artificial control 5′ UTR (Luc-Control) in anon-ischemic kidney model. FIG. 7F is a graph showing quantification ofthe experiment described in FIG. 7E (n=6). FIG. 7G is a schematicdiagram illustrating the experimental protocol used to evaluate thetranslation efficiency of Luc modRNA comprising the 5′ UTR of Ces1d(Luc-Ces1d) or an artificial control 5′ UTR (Luc-Control) in an ischemickidney model. FIG. 7H is a graph showing quantification of theexperiment described in FIG. 7G (n=6). Two-way ANOVA, Tukey's MultipleComparison Test for FIGS. 7B, 7D, 7F, and 7H. *, P<0.05, N.S., NotSignificant.

FIGS. 8A-8E demonstrate that a specific RNA element in the 5′ UTR ofCes1d significantly enhances Luc modRNA translation in an ischemic heartmouse model. FIG. 8A is a table listing elements of the 5′ UTR of Ces1dthat have been conserved among various species (Elements A-E). FIG. 8Bis a schematic diagram illustrating the experimental protocol used toevaluate the translation efficiency of Luc modRNA constructs comprisingthe RNA elements identified in FIG. 8A (i.e., Luc-Element A, Luc-ElementB, Luc-Element C, Luc-Element D, and Luc-Element E) as well as a LucmodRNA construct comprising the 5′ UTR of Ces1d (Luc-Ces1d) in neonatalrat CMs using IVIS® analysis. FIG. 8C is a graph showing quantificationof the experiment described in FIG. 8B (n=4). FIG. 8D is a schematicdiagram illustrating the experimental protocol used to evaluate thetranslation efficiency of Luc-modRNA constructs comprising an artificialcontrol 5′ UTR (Luc-Control), the full-length 5′ UTR of Ces1d(Luc-Ces1d), or Element D of the 5′ UTR of Ces1d (Luc-Element D) in anischemic heart model. FIG. 8E is a graph showing quantification of theexperiment described in FIG. 8D (n=5). One-way ANOVA, Tukey's MultipleComparison Test for FIG. 8C. Two-way ANOVA, Tukey's Multiple ComparisonTest for FIG. 8E. ***, P<0.001, **, P<0.01, *, P<0.05, N.S., NotSignificant.

FIGS. 9A-9B demonstrate that Element D of the 5′ UTR of Ces1d does notincrease mRNA translation significantly more than the full-length 5′ UTRof Ces1d in a liver ischemic mouse model. FIG. 9A is a schematic diagramillustrating the experimental protocol used to evaluate the translationefficiency of Luc modRNA comprising the full-length 5′ UTR of Ces1d orElement D of the 5′ UTR of Ces1d in an ischemic liver mouse model. FIG.9B is a graph showing quantification of the experiments described inFIG. 9A (n=4). Two-way ANOVA, Tukey's Multiple Comparison Test. N.S.,Not Significant.

FIGS. 10A-10B demonstrate that Element D in the 5′ UTR of Ces1d does notsignificantly increase mRNA translation over the full length 5′ UTR ofCes1d or control 5′ UTR in a heart non-ischemic mouse model. FIG. 10A isa schematic diagram illustrating the experimental protocol used toevaluate the translation efficiency of Luc modRNA carrying the fulllength 5′ UTR of Ces1d or the control 5′ UTR insert in a heartnon-ischemic mouse model. FIG. 10B is a graph showing quantification ofthe experiment described in FIG. 10A using IVIS® analysis. Two-wayANOVA, Turkey's Multiple Comparison Test. N.S., not significant.

FIG. 11 is a flow diagram illustrating one embodiment of a method ofidentifying a 5′ untranslated region (5′ UTR) for selectively enhancingtranslation of a heterologous protein of interest in a target cell ortissue. Step A corresponds to obtaining a disease sample (i.e., a firstsample of living tissue comprising a target cell under diseaseconditions) and a reference sample (i.e., second sample of living tissuecomprising the target cell under non-disease conditions); step Bcorresponds to proteome analysis (i.e., quantifying genes that aretranslated) and transcriptome analysis (i.e., quantifying genes that aretranscribed) in the disease and reference samples (i.e., the first andsecond samples); step C corresponds to identifying genes which (i) aretranscribed at similar or lower levels in the first sample relative tothe second sample and (ii) are translated at higher levels in the firstsample relative to the second sample; and step D corresponds toidentifying the 5′ UTR of the identified genes, where the identified 5′UTR is capable of selectively enhancing translation of a heterologousprotein of interest in a target cell or tissue.

DETAILED DESCRIPTION

The present application relates to the identification of nucleic acidregions that enhance the translation efficiency of a target protein.Disclosed herein are nucleic acid molecules comprising at least aportion of a 5′ untranslated region (5′ UTR) of an identified gene andpharmaceutical compositions comprising the same.

As used herein, the singular forms “a”, “an”, and “the” include pluralreference unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs.

A first aspect of the present application relates to a nucleic acidmolecule comprising a first nucleic acid sequence comprising at least aportion of a 5′ untranslated region (5′ UTR) of a carboxylesterase geneand a second nucleic acid sequence encoding a protein of interest, wherethe second nucleic acid sequence is heterologous to and operativelycoupled to the first nucleic acid sequence.

As used herein, the term “operably coupled” refers to the sequential andfunction arrangement between a 5′ UTR and a nucleic acid encoding aprotein of interest, where the 5′ UTR modulates translation of a nucleicacid sequence encoding a protein of interest.

As used herein, the term “nucleoside” refers to a molecule comprising anitrogenous base (i.e., a nucleobase) linked to a pentose (e.g.,deoxyribose or ribose) sugar. Typical nitrogenous bases which formnucleosides include adenine, guanine, cytosine, 5-methyl cytosine,uracil, and thymine. Suitable ribonucleosides (which comprise ribose asthe pentose sugar) include, e.g., adenosine (A), guanosine (G),5-methyluridine (m⁵U), uridine (U), and cytidine (C).

As used herein, the term “nucleotide” refers to a molecule comprising anucleoside (e.g., a ribonucleoside) and a phosphate group.Ribonucleotides include, e.g., adenosine monophosphate, adenosinediphosphate, adenosine triphosphate, guanosine monophosphate, guanosinediphosphate, guanosine triphosphate, cytidine monophosphate, cytidinediphosphate, cytidine triphosphate, uridine monophosphate, uridinediphosphate, uridine triphosphate, and derivatives thereof.

As used herein, the term “messenger RNA” (also, mRNA) refers to aribonucleotide sequence that encodes a protein of interest and can betranslated to produce the encoded protein of interest in vitro, in vivo,in situ, or ex vivo.

As used herein, the term “modified” or “mod” in reference to RNA refersto an alteration of a ribonucleotide that can be, for example,incorporated into an mRNA molecule. Modifications to an mRNA moleculecan include, for example and without limitation, physical or chemicalmodifications to a base, such as, for example and without limitation,the depletion of a base or a chemical modification of a base (see, e.g.,U.S. Pat. No. 8,278,036 to Kariko et al.; U.S. Pat. No. 10,086,043 toChien et al.; and U.S. Patent Application Publication No. 2019/0203226to Zangi et al., which are hereby incorporated by reference in theirentirety).

In some embodiments, the first and second nucleic acids are ribonucleicacids. In certain embodiments, the first and second nucleic acids aremRNAs. In certain other embodiments, the first and second nucleic acidsare modified mRNAs (modRNAs).

modRNAs can be prepared as described in, for example, U.S. Pat. No.8,278,036 to Kariko et al.; Sultana et al., “Optimizing Cardiac Deliveryof Modified mRNA,” Mol. Ther. 25(6):1306-1315 (2017); and Hadas et al.,“Optimizing Modified mRNA In Vitro Synthesis Protocol for Heart GeneTherapy,” Mol. Ther. Methods Clin. Dev. 14(13):300-305 (2019), which arehereby incorporated by reference in their entirety. In some embodiments,modRNA is generated by in vitro transcription. The modRNA may be invitro transcribed, e.g., from a linear DNA template using one or morereagents selected from the group consisting of a cap analog, guanosinetriphosphate, adenosine triphosphate, cytidine triphosphate, uridinetriphosphate, and derivatives thereof (Hadas et al., “OptimizingModified mRNA In Vitro Synthesis Protocol for Heart Gene Therapy,” Mol.Ther. Methods Clin. Dev. 14(13):300-305 (2019), which is herebyincorporated by reference in its entirety).

The cap analog may be selected from Anti-Reverse Cap Analog (ARCA)3′-O-Me-m⁷G(5′)ppp(5′)G (Hadas et al., “Optimizing Modified mRNA InVitro Synthesis Protocol for Heart Gene Therapy,” Mol. Ther. MethodsClin. Dev. 14(13):300-305 (2019), which is hereby incorporated byreference in its entirety), standard cap analog m⁷G(5′)ppp(5′)G,unmethylated cap analog G(5′)ppp(5′)G, methylated cap analog for A+1sites m⁷G(5′)ppp(5′)A, and unmethylated cap analog for A+1 sitesG(5′)ppp(5′)A. In certain embodiments, the cap analog is Anti-ReverseCap Analog (ARCA) 3′-O-Me-m⁷G(5′)ppp(5′)G.

Suitable derivatives of guanosine triphosphate, adenosine triphosphate,cytidine triphosphate, and uridine triphosphate are well known in theart and include, e.g., modifications to the ribonucleoside.Ribonucleosides can, for example, be modified to produce modRNAs having,e.g., increased stability and/or clearance in tissues, improved receptoruptake and/or kinetics, improved cellular access by the nucleic acidmolecules, improved engagement with translational machinery, improvedmRNA half-life, increased translation efficiency, improved immuneevasion, improved protein production capacity, improved secretionefficiency, improved accessibility to circulation, improved proteinhalf-life and/or modulation of a cell's status, improved function,improved activity, or for any other reason.

According to some embodiments, modRNA is in vitro transcribed from aplasmid template using one or more reagents selected from the groupconsisting of 3′-O-Me-m7G(5′)ppp(5′)G, guanosine triphosphate, adenosinetriphosphate, cytidine triphosphate, andN1-methylpseudouridine-5-triphosphate. Thus, in certain embodiments ofthe invention disclosed herein, the modRNAs compriseN1-methylpseudouridine. In other embodiments, the modRNAs comprisepseudouridine or methylpseudouridine.

Additional suitable modifications to a modRNA or mRNA molecule are wellknown in the art (see, e.g., U U.S. Pat. No. 8,278,036 to Kariko et al.;U.S. Pat. No. 10,086,043 to Chien et al.; U.S. Patent ApplicationPublication No. 2019/0203226 to Zangi et al.; and U.S. PatentApplication Publication No. 2018/0353618 to Burkhardt et al., which arehereby incorporated by reference in their entirety). In someembodiments, the nucleoside that is modified in the modRNA is a uridine(U), a cytidine (C), an adenine (A), or guanine (G). The modifiednucleoside can be, for example, m⁵C (5-methylcytidine), m⁶A(N⁶-methyladenosine), s²U (2-thiouridien), ψ (pseudouridine), or Um(2-O-methyluridine). Some exemplary chemical modifications ofnucleosides in the modRNA molecule may further include, for example andwithout limitation, pyridine-4-one ribonucleoside, 5-aza-uridine,2-thio-5-aza uridine, 2-thiouridine, 4-thio pseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl pseudouridine, 5-propynyl uridine, 1-propynylpseudouridine, 5-taurinomethyluridine, 1-taurinomethyl pseudouridine,5-taurinomethyl-2-thio uridine, 1-taurinomethyl-4-thio uridine, 5-methyluridine, 1-methyl pseudouridine, 4-thio-1-methyl pseudouridine,2-thio-1-methyl pseudouridine, 1-methyl-1-deaza pseudouridine,2-thio-1-methyl-1-deaza pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio dihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio uridine,4-methoxy pseudouridine, 4-methoxy-2-thio pseudouridine, 5-aza cytidine,pseudoisocytidine, 3-methyl cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methyl cytidine, 4-thio pseudoisocytidine,4-thio-1-methyl pseudoisocytidine, 4-thio-1-methyl-1-deazapseudoisocytidine, 1-methyl-1-deaza pseudoisocytidine, zebularine, 5-azazebularine, 5-methyl zebularine, 5-aza-2-thio zebularine, 2-thiozebularine, 2-methoxy cytidine, 2-methoxy-5-methyl cytidine, 4-methoxypseudoisocytidine, 4-methoxy-1-methyl pseudoisocytidine, 2-aminopurine,2,6-diaminopurine, 7-deaza adenine, 7-deaza-8-aza adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N⁶-methyladenosine, N⁶-isopentenyladenosine,N⁶-(cis-hydroxyisopentenyl) adenosine,2-methylthio-N⁶-(cis-hydroxyisopentenyl) adenosine,N⁶-glycinylcarbamoyladenosine, N⁶-threonylcarb amoyladenosine,2-methylthio-N⁶-threonyl carbamoyladenosine, N⁶,N⁶-dimethyladenosine,7-methyladenine, 2-methylthio adenine, 2-methoxy adenine, inosine,1-methyl inosine, wyosine, wybutosine, 7-deaza guanosine, 7-deaza-8-azaguanosine, 6-thio guanosine, 6-thio-7-deaza guanosine,6-thio-7-deaza-8-aza guanosine, 7-methyl guanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxy guanosine, 1-methylguanosine,N²-methylguanosine, N²,N²-dimethylguanosine, 8-oxo guanosine,7-methyl-8-oxo guanosine, 1-methyl-6-thio guanosine, N²-methyl-6-thioguanosine, or N²,N²-dimethyl-6-thio guanosine.

In one embodiment, the modifications made to the modRNA areindependently selected from the group consisting of 5-methylcytosine,pseudouridine, and 1-methylpseudouridine.

In some embodiments, the modRNA comprises a modified uracil selectedfrom the group consisting of pseudouridine (w), pyridine-4-oneribonucleoside, 5-aza uridine, 6-aza uridine, 2-thio-5-aza uridine,2-thio uridine (s2U), 4-thio uridine (s4U), 4-thio pseudouridine, 2-thiopseudouridine, 5-hydroxy uridine (ho⁵U), 5-aminoallyl uridine, 5-halouridine (e.g., 5-iodom uridine or 5-bromo uridine), 3-methyl uridine(m³U), 5-methoxy uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U),uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl uridine(cm⁵U), 1-carboxymethyl pseudouridine, 5-carboxyhydroxymethyl uridine(chm⁵U), 5-carboxyhydroxym ethyl uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl uridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s2U), 5-aminomethyl-2-thio uridine (nm⁵s2U),5-methylaminomethyl uridine (mnm⁵U), 5-methylaminomethyl-2-thio uridine(mnm⁵s2U), 5-methylaminomethyl-2-seleno uridine (mnm⁵se²U),5-carbamoylmethyl uridine (ncm⁵U), 5-carboxymethylaminomethyl uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio uridine (cmnm⁵s2U),5-propynyl uridine, 1-propynyl pseudouridine, 5-taurinomethyl uridine(Tcm⁵U), 1-taurinomethyl pseudouridine, 5-taurinomethyl-2-thio uridine(™⁵s2U), 1-taurinomethyl-4-thio pseudouridine, 5-methyl uridine (m⁵U,e.g., having the nucleobase deoxythymine), 1-methyl pseudouridine (m¹ψ),5-methyl-2-thio uridine (m⁵s2U), 1-methyl-4-thio pseudouridine (m₁s⁴ψ),4-thio-1-methyl pseudouridine, 3-methyl pseudouridine (m³ψ),2-thio-1-methyl pseudouridine, 1-methyl-1-deaza pseudouridine,2-thio-1-methyl-1-deaza pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl dihydrouridine (m⁵D),2-thio dihydrouridine, 2-thio dihydropseudouridine, 2-methoxy uridine,2-methoxy-4-thio uridine, 4-methoxy pseudouridine, 4-methoxy-2-thiopseudouridine, N¹-methyl pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp³U), 1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine(acp³ψ), 5-(isopentenylaminomethyl) uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio uridine (inm⁵s2U), α-thio uridine,2′-O-methyl uridine (Um), 5,2′-O-dimethyl uridine (m⁵Um), 2′-O-methylpseudouridine (vm), 2-thio-2′-O-methyl uridine (s2Um),5-methoxycarbonylmethyl-2′-O-methyl uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl uridine (cmnm⁵Um),3,2′-O-dimethyl uridine (m³Um), 5-(isopentenylaminomethyl)-2′-O-methyluridine (inm⁵Um), 1-thio uridine, deoxythymidine, 2′-F-ara uridine, 2′-Furidine, 2′-OH-ara uridine, 5-(2-carbomethoxyvinyl) uridine, and5-3-(1-E-propenylamino) uridine.

In some embodiments, the modRNA comprises a modified cytosine selectedfrom the group consisting of 5-aza cytidine, 6-aza cytidine,pseudoisocytidine, 3-methyl cytidine (m³C), N⁴-acetyl cytidine (act),5-formyl cytidine (f⁵C), N⁴-methyl cytidine (m⁴C), 5-methyl cytidine(m⁵C), 5-halo cytidine (e.g., 5-iodo cytidine), 5-hydroxymethyl cytidine(hm⁵C), 1-methyl pseudoisocytidine, pyrrolo-cytidine,pyrrolo-pseudoisocytidine, 2-thio cytidine (s2C), 2-thio-5-methylcytidine, 4-thio pseudoisocytidine, 4-thio-1-methyl pseudoisocytidine,4-thio-1-methyl-1-deaza pseudoisocytidine, 1-methyl-1-deazapseudoisocytidine, zebularine, 5-aza zebularine, 5-methyl zebularine,5-aza-2-thio zebularine, 2-thio zebularine, 2-methoxy cytidine,2-methoxy-5-methyl cytidine, 4-methoxy pseudoisocytidine,4-methoxy-1-methyl pseudoisocytidine, lysidine (k²C), alpha-thiocytidine, 2′-O-methyl cytidine (Cm), 5,2′-O-dimethyl cytidine (m⁵Cm),N⁴-acetyl-2′-O-methyl cytidine (ac⁴Cm), N⁴,2′-O-dimethyl cytidine(m⁴Cm), 5-formyl-2′-O-methyl cytidine (f⁵Cm), N⁴,N⁴,2′-O-trimethylcytidine (m⁴ ₂Cm), 1-thio cytidine, 2′-F-ara cytidine, 2′-F cytidine,and 2′-OH-ara cytidine.

In some embodiments, the modRNA comprises a modified adenine selectedfrom the group consisting of 2-amino purine, 2,6-diamino purine,2-amino-6-halo purine (e.g., 2-amino-6-chloro purine), 6-halo purine(e.g., 6-chloro purine), 2-amino-6-methyl purine, 8-azido adenosine,7-deaza adenine, 7-deaza-8-aza adenine, 7-deaza-2-amino purine,7-deaza-8-aza-2-amino purine, 7-deaza-2,6-diamino purine,7-deaza-8-aza-2,6-diamino purine, 1-methyl adenosine (m′A), 2-methyladenine (m²A), N⁶-methyl adenosine (m⁶A), 2-methylthio-N⁶-methyladenosine (ms2m⁶A), N⁶-isopentenyl adenosine (i⁶A),2-methylthio-N⁶-isopentenyl adenosine (ms²i⁶A),N⁶-(cis-hydroxyisopentenyl) adenosine (io⁶A),2-methylthio-N⁶-(cis-hydroxyisopentenyl) adenosine (ms²io⁶A), N⁶glycinylcarbamoyl adenosine (g⁶A), N⁶-threonylcarbamoyl adenosine (t⁶A),N⁶-methyl-N⁶-threonylcarbamoyl adenosine (m⁶ ₂A),2-methylthio-N⁶-threonylcarbamoyl adenosine (ms²g⁶A), N⁶,N⁶-dimethyladenosine (m⁶ ₂A), N⁶-hydroxynorvalylcarbamoyl adenosine (hn⁶A),2-methylthio-N⁶-hydroxynorvalylcarbamoyl adenosine (ms²hn⁶A), N⁶-acetyladenosine (ac⁶A), 7-methyl adenine, 2-methylthio adenine, 2-methoxyadenine, alpha-thio adenosine, 2′-O-methyl adenosine (Am),N⁶,2′-O-dimethyl adenosine (m⁶Am) N⁶,N⁶,2′-O-trimethyl adenosine (m⁶₂Am), 1,2′-O-dimethyl adenosine (m′Am), 2′-O-ribosyl adenosine(phosphate) (Ar(p)), 2-amino-N⁶-methyl purine, 1-thio adenosine, 8-azidoadenosine, 2′-F-ara adenosine, 2′-F adenosine, 2′-OH-ara adenosine, andN⁶-(19-amino-pentaoxanonadecyl) adenosine.

In some embodiments, the modRNA comprises a modified guanine selectedfrom the group consisting of inosine (I), 1-methyl inosine (m′I),wyosine (imG), methylwyosine (mimG), 4-demethyl wyosine (imG-14),isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW),hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyWy),7-deaza guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosylqueuosine (galQ), mannosyl queuosine (manQ), 7-cyano-7-deaza guanosine(preQ₀), 7-aminomethyl-7-deaza guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza guanosine, 6-thio guanosine, 6-thio-7-deaza guanosine,6-thio-7-deaza-8-aza guanosine, 7-methyl guanosine (m⁷G),6-thio-7-methyl guanosine, 7-methyl inosine, 6-methoxy guanosine,1-methyl guanosine (m¹G), N²-methyl-guanosine (m²G), N²,N²-dimethylguanosine (m² ₂G), N^(2,7)-dimethyl guanosine (m^(2,7)G), N²,N^(2,7)-dimethyl guanosine (m^(2,2,7)G), 8-oxo guanosine, 7-methyl-8-oxoguanosine, 1-methio guanosine, N²-methyl-6-thio guanosine,N²,N²-dimethyl-6-thio guanosine, alpha-thio guanosine, 2′-O-methylguanosine (Gm), N²-methyl-2′-O-methyl guanosine (m²Gm),N²,N²-dimethyl-2′-O-methyl guanosine (m² ₂Gm), 1-methyl-2′-O-methylguanosine (m¹Gm), N^(2,7)-dimethyl-2′-O-methyl guanosine (m^(2,7)Gm),2′-O-methyl inosine (1m), 1,2′-O-dimethyl inosine (m¹Gm), 2′-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio guanosine, O⁶-methyl guanosine,2′-F-ara guanosine, and 2′-F guanosine.

modRNA may include, for example, a non-natural or modified nucleotide.The non-natural or modified nucleotide may include, for example, abackbone modification, sugar modification, or base modification. Thenon-natural or modified nucleotide may include, for example, a basemodification. In some embodiments, the base modification is selectedfrom the group consisting of 2-amino-6-chloropurine riboside 5′triphosphate, 2-aminoadenosine 5′ triphosphate, 2-thiocytidine 5′triphosphate, 2-thiouridine 5′ triphosphate, 4-thiouridine 5′triphosphate, 5-aminoallylcytidine 5′ triphosphate, 5-aminoallyluridine5′ triphosphate, 5-bromocytidine 5′ triphosphate, 5-bromouridine 5′triphosphate, 5-iodocytidine 5′ triphosphate, 5-iodouridine 5′triphosphate, 5-methylcytidine 5′ triphosphate, 5-methyluridine 5′triphosphate, 6-azacytidine 5′ triphosphate, 6-azauridine 5′triphosphate, 6-chloropurine riboside 5′-triphosphate, 7-deazaadenosine5′ triphosphate, 7-deazaguanosine 5′ triphosphate, 8-azaadenosine 5′triphosphate, 8-azidoadenosine 5′ triphosphate, benzimidazole riboside5′ triphosphate, N¹-methyladenosine 5′ triphosphate, N¹-methylguanosine5′ triphosphate, N⁶-methyladenosine 5′ triphosphate, 0⁶-methylguanosine5′ triphosphate, N¹-methyl-pseudouridine 5′ triphosphate, puromycin5′-triphosphate, and xanthosine 5′ triphosphate. Thus, according to someembodiments, the modRNA comprises N¹-methyl-pseudouridine 5′triphosphate.

Other modifications include, for example, those described in Tavernieret al., “mRNA as Gene Therapeutic: How to Control Protein Expression,”J. Control. Release 150(3):238-247 (2011); Anderson et al., “NucleosideModifications in RNA Limit Activation of 2′-5′-Oligoadenylate Synthetaseand Increase Resistance to Cleavage by RNase L,” Nucleic Acids Res.39(21):9329-9338 (2011); Kormann et al., “Expression of TherapeuticProteins After Delivery of Chemically Modified mRNA in Mice,” Nat.Biotechnol. 29(2):154-157 (2011); Kariko et al., “Incorporation ofPseudouridine into mRNA Yields Superior Nonimmunogenic Vector withIncreased Translational Capacity and Biological Stability,” Mol. Ther.16(11):1833-1840 (2008); Kariko et al., “Suppression of RNA Recognitionby Toll-Like Receptors: The Impact of Nucleoside Modification and theEvolutionary Origin of RNA,” Immunity 23(2):165-175 (2005); and Warrenet al., “Highly Efficient Reprogramming to Pluripotency and DirectedDifferentiation of Human Cells with Synthetic Modified mRNA,” Cell StemCell 7(5):618-630 (2010), which are hereby incorporated by reference intheir entirety.

In some embodiments, the modRNA comprises a modified nucleoside selectedfrom the group consisting of m⁵C, m⁵U, m⁶A, s²U, Ψ, or 2′-O-methyl-U.

As used herein, the term “untranslated region” or “UTR” refers to atranscribed but untranslated region of a mRNA molecule. The 5′ UTRstarts at the transcription start site and continues to the start codonbut does not include the start codon; whereas, the 3′ UTR startsimmediately following the stop codon and continues until thetranscriptional termination signal. Natural 5′ UTRs help translationinitiation (Ong et al., “The Role of 5′ Untranslated Region inTranslational Suppression of OKL38 mRNA in Hepatocellular Carcinoma,”Oncogene 26(8):1155-65 (2007); Leppek et al., “Functional 5′ UTR mRNAStructures in Eukaryotic Translation Regulation and How to Find Them,”Nat. Rev. Mol. Cell Biol. 19(3):158-174 (2018); and van der Velden &Thomas, “The Role of the 5′ Untranslated Region of an mRNA inTranslation Regulation During Development,” Int. J. Biochem. Cell Biol.31(1):87-106 (1999), which are hereby incorporated by reference in theirentirety), and may comprise features such as, e.g., Kozak sequences,which facilitate translation initiation by the ribosome for many genes.

As demonstrated by the Examples below, modRNA constructs comprising the5′ UTR of, e.g., a mammalian carboxylesterase gene enhances thetranslation efficiency of a protein of interest as compared to modRNAconstructs comprising a reference (i.e., an artificial control) 5′ UTRsequence. Hence, in a first aspect, the present application relates to anucleic acid molecule comprising a first nucleic acid sequencecomprising at least a portion of a 5′ UTR of a carboxylesterase gene anda second nucleic acid sequence encoding a protein of interest, where thesecond nucleic acid sequence is heterologous to and operatively coupledto the first nucleic acid sequence.

According to some embodiments, the carboxylesterase gene is a mammaliancarboxylesterase gene. Suitable mammalian carboxylesterase genes andtheir corresponding 5′ UTR sequences (i.e., SEQ ID NO:1 (mouse), SEQ IDNO:2 (rat), SEQ ID NO:3 (pig), SEQ ID NO:4 (gerbil), SEQ ID NO:5(human), and SEQ ID NO:6 (monkey)) are shown in Table 1 below.

TABLE 1 Mammalian Carboxylesterase Genes SEQ ID Name Species5′ UTR Sequence^(†) NO: carboxyl- MusAGGAGGCGGGTCCCCTGGTCCACAACAGAAGCATT 1 esterase musculusGCTAAAGCAGCAGATAGC

1D(Ces1d) (Mouse)

TTGTCCTTCCACA (GenBank Accession No. NM_053200.2, positions 1-85) RattusTGCTAAAGGAACAAATAGC

2 norvegicus

TTGTCCTTCCACA (Rat) (GenBank Accession No. NM_133295.3, positions 1-51)Cavia GAATTCACAGGATCATATCCAGTACTGTTCAAGGA 3 porcellusCAAGTGCATTTCCATGAATCAGGACAGAGAGC

(Pig)

TGTTGTCTTCCCATG (GenBank Accession No. NM_001173109.1, positions 1-98)Meriones CAGGACCTTGGGTCCACAACAGCATTGCTAAAGCA 4 unguiculatus GCAGATA

TTGTCCT (Gerbil) TCCACA (GenBank Accession No.XM_021659724.1, positions 1-76) carboxyl- Homo sapiensAGCGCAGGGCGGTAACTCTGGGCGGGGCTGGGCTC 5 esterase (Human)CAGGGCTGGACAGCACAGTCCCTCTGAACTGCACA 1(Ces1) GAGACCTCGC

TGTCGCCCTTCC ACG (GenBank Accession No. NG_012057.1,positions 5001-5108) Macaca GGCTTTACTGCTATCTCCCAATTAGAGGATTAGGC 6mulatto AATTGGCAGCTCAGGGTGGTAACTCAGGGCCTGG (Rhesus(GenBank Accession No. monkey) XM_015126191.2, positions 1-69) ^(†)RNAElement D of the carboxylesterase 5′ UTR sequences are shown in boldunderline in the above Table 1.

In some embodiments, the carboxylesterase gene is a carboxylesterase 1D(Ces1d) gene. The Ces1d gene may be a murine Ces1d gene. In accordancewith such embodiments, the first nucleic acid sequence comprises atleast a portion of the nucleic acid sequence of SEQ ID NO:1. Additionalsuitable Ces1d gene sequences are shown in Table 1 supra.

In other embodiments, the carboxylase gene is a carboxylesterase 1(CES1) gene. The CES1 gene may be a human CES1 gene. In accordance withsuch embodiments, the first nucleic acid sequence comprises at least aportion of the nucleic acid sequence of SEQ ID NO:5. Additional suitableCES1 gene sequences are shown in Table 1 supra.

The Examples below demonstrate the ability of modRNA constructscomprising RNA Element D of the 5′ UTR of a mammalian carboxylesterasegene to enhance the translation efficiency of a protein of interest ascompared to modRNA constructs comprising an artificial control 5′ UTRsequence. In some embodiments, the RNA Element D corresponds topositions 54-72 of SEQ ID NO:1. Thus, in certain embodiments, the firstnucleic acid sequence comprises SEQ ID NO:10 (i.e., RNA Element D of the5′ UTR of Mouse Ces1d). In select embodiments, the first nucleic acidsequence comprises SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:24.

In other embodiments, the carboxylesterase gene is human CES1 and thefirst nucleic acid sequence comprises nucleotides 81-93 of SEQ ID NO:5(i.e., RNA Element D of the 5′ UTR of Human CES1). Thus, in certainembodiments, the RNA Element D corresponds to SEQ ID NO:29 (i.e., RNAElement D of the 5′ UTR of Human Ces1d).

According to some embodiments, the second nucleic acid sequence encodesa protein of interest. Suitable proteins of interest, which are encodedby the second nucleic acid sequence include, for example and withoutlimitation, a therapeutic protein or a reporter protein.

When the second nucleic acid sequence encodes a therapeutic protein, thetherapeutic protein may be, according to one embodiment, a cell cycleinducer. Suitable cell cycle inducers include, without limitation,Lin28, Pyruvate Kinase Muscle Isozyme M2 (Pkm2), β-catenin, caERBB2, YesAssociated Protein 1 (YAP), Cyclin D1, and c-Myc.

Lin28 is a known suppressor of Let7 that tightly controls cell cycleregulators (D′Uva et al., “ERBB2 Triggers Mammalian Heart Regenerationby Promoting Cardiomyocyte Dedifferentiation and Proliferation,” Nat.Cell Biol. 17(5):627-638 (2015); Engel et al., “p38 MAP KinaseInhibition Enables Proliferation of Adult Mammalian Cardiomyocytes,”Genes Dev. 19(10):1175-1187 (2005); Lee et al., “Cell Cycle Re-Entry andMitochondrial Defects in Myc-Mediated Hypertrophic Cardiomyopathy andHeart Failure,” PloS One 4(9):e7172 (2009); Liao et al.,“Cardiac-Specific Overexpression of Cyclin-Dependent Kinase 2 IncreasesSmaller Mononuclear Cardiomyocytes,” Circ. Res. 88(4):443-450 (2001);Ozhan & Weidinger, “Wnt/β-Catenin Signaling in Heart Regeneration,” CellRegen. 4(1):3 (2015), which are hereby incorporated by reference intheir entirety). Treatment of cardiomyocytes post-myocardial infarctionusing modRNA constructs encoding Lin28 has been shown to inducecardiomyocyte proliferation, reduce apoptosis, and increase capillarydensity (see, e.g., U.S. Patent Application Publication No. 2019/0203226to Zangi et al., which is hereby incorporated by reference in itsentirety). In some embodiments, the cell cycle inducer is Lin28.

Pyruvate Kinase Muscle Isozyme M2 (Pkm2) is a pro-proliferative factor,highly expressed in regenerative fetal and early neonatalcardiomyocytes, but not in adult cardiomyocytes (see, e.g., U.S. PatentApplication Publication No. 2019/0203226 to Zangi et al., which ishereby incorporated by reference in its entirety). In the cytoplasm,Pkm2 shifts the metabolic fate from glycolysis to pentose phosphatepathway (“PPP”) by reducing the conversion of phosphoenolpyruvate topyruvate (Dong et al., “PKM2 and Cancer: The Function of PKM2 BeyondGlycolysis,” Oncol. Lett. 11(3):1980-1986 (2016) and Riganti et al.,“The Pentose Phosphate Pathway: An Antioxidant Defense and a Crossroadin Tumor Cell Fate,” Free Rad. Biol. Med. 53(3):421-436 (2012), whichare hereby incorporated by reference in their entirety), which leads tothe accumulation of galactose, a glycolysis intermediate, and activationof PPP via Glucose-6-phosphate dehydrogenase (G6pd) (Kumar et al.,“Moderate DNA Damage Promotes Metabolic Flux into PPP via PKM2 Y-105Phosphorylation: A feature that Favours Cancer Cells,” Mol. Biol. Rep.42(8):1317-1321 (2015); Salani et al., “IGF1 Regulates PKM2 FunctionThrough Akt Phosphorylation,” Cell Cycle 14(10):1559-1567 (2015); andWong et al., “PKM2, a Central Point of Regulation in Cancer Metabolism,”Int. J. Cell Biol. 2013:242513 (2013), which are hereby incorporated byreference in their entirety). The PPP pathway activation leads to thesynthesis of nucleotides, amino acids, and lipids and the production ofreduced NADPH, increase nitric oxide synthase and DNA repair (Luo &Semenza, “Pyruvate Kinase M2 Regulates Glucose Metabolism by Functioningas a Coactivator for Hypoxia-Inducible Factor 1 in Cancer Cells,”Oncotarget 2(7):551-556 (2011); Mazurek, “Pyruvate Kinase Type M2: A KeyRegulator of the Metabolic Budget System in Tumor Cells,” Int. J.Biochem. Cell Biol. 43(7):969-980 (2011); Vander Heiden et al.,“Understanding the Warburg Effect: The Metabolic Requirements of CellProliferation,” Science 324(5930):1029-1033 (2009); Luo et al.,“Induction of Apoptosis in Human Leukemic Cell Lines by DiallylDisulfide via Modulation of EGFR/ERK/PKM2 Signaling Pathways,” AsianPac. J. Cancer Prev. 16(8):3509-3515 (2015); Zhang et al., “NuclearTranslocation of PKM2 Modulates Astrocyte Proliferation via p27 andβ-Catenin Pathway After Spinal Cord Injury,” Cell Cycle 14(16):2609-2618(2015); and David et al., “HnRNP Proteins Controlled by c-Myc DeregulatePyruvate Kinase mRNA Splicing in Cancer,” Nature 463(7279):364-368(2010), which are hereby incorporated by reference in their entirety).In the nucleus, Pkm2 directly interacts with the transcription factorsμ-catenin and Hif1α. This interaction promotes the expression of genessuch as in Ccdn1, c-Myc and Vegfa, and Bc12 (Luo et al., “PyruvateKinase M2 is a PHD3-Stimulated Coactivator for Hypoxia-Inducible Factor1,” Cell 145(5):732-744 (2011) and Azoitei et al., “PKM2 Promotes TumorAngiogenesis by Regulating HIF-1alpha Through NF-kappaB Activation,”Mol. Cancer 15:3 (2016), which are hereby incorporated by reference intheir entirety). Restoration of Pkm2 levels using modRNA into adultcardiomyocytes post-myocardial infarction has been shown tosignificantly and exclusively induce cardiomyocyte proliferation;associate with improved cardiac function, reduced scar size, andincreased heart to body weight ratio; reduce cardiomyocyte size; reduceapoptosis; and increase capillary density (see, e.g., U.S. PatentApplication Publication No. 2019/0203226 to Zangi et al., which ishereby incorporated by reference in its entirety). In some embodiments,the cell cycle inducer is Pyruvate Kinase Muscle Isozyme M2 (Pkm2).

β-catenin is a subunit of the cadherin protein complex and acts as anintracellular signal transducer in the Wnt signaling pathway. In cardiacmuscle, β-catenin localizes to adherens junctions in intercalated discstructures, which are critical for electrical and mechanical couplingbetween adjacent cardiomyocytes. Loss of β-catenin during early heartformation results in multiple heart defects and lethality demonstratingits crucial function for embryonic heart development (Lickert et al.,“Formation of Multiple Hearts in Mice Following Deletion of Beta-Cateninin the Embryonic Endoderm,” Dev. Cell 3:171-181 (2002), which is herebyincorporated by reference in its entirety). In adults, β-cateninsignaling plays an important role in normal and stress-induced cardiachypertrophic remodeling (Chen et al., “The Beta-Catenin/T-cellFactor/Lymphocyte Enhancer Factor Signaling Pathway is Required forNormal and Stress-Induced Cardiac Hypertrophy,” Mol. Cell Biol.26:4462-4473 (2006), which is hereby incorporated by reference in itsentirety). Wnt/β-catenin signaling may function in a stage-specificbiphasic manner, either promoting or inhibiting cardiogenesis(Stubenvoll et al., “Attenuation of Wnt/β-catenin Activity ReversesEnhanced Generation of Cardiomyocytes and Cardiac Defects Caused by theloss of Emerin,” Human Mol. Gen. 24(3):802-813 (2015) and Grigoryan etal., “Deciphering the Function of Canonical Wnt Signals in Developmentand Disease: Conditional Loss- and Gain-of-Function Mutations ofBeta-Catenin in Mice,” Genes Dev. 22:2308-2341 (2008), which is herebyincorporated by reference in its entirety).

ERBB2 (erb-b2 receptor tyrosine kinase 2) forms a heterodimer with otherepidermal growth factor receptor tyrosine kinase family members. ERBB2is required for cardiomyocyte proliferation at embryonic/neonatal stages(D′Uva et al., “ERBB2 Triggers Mammalian Heart Regeneration by PromotingCardiomyocyte Dedifferentiation and Proliferation,” Nat. Cell Biol.17(5):627-638 (2015), which is hereby incorporated by reference in itsentirety). Transient induction of a constitutively active ERBB2(caERBB2) for 10-20 days after ischemic injury, either in juvenile oradult hearts, has been shown to trigger a series of events starting withcardiomyocyte dedifferentiation, proliferation, neovascularization and,after ERBB2-signaling termination, proceeding to cardiomyocytere-differentiation that together lead to anatomical and functional heartregeneration (D′Uva et al., “ERBB2 Triggers Mammalian Heart Regenerationby Promoting Cardiomyocyte Dedifferentiation and Proliferation,” Nat.Cell Biol. 17(5):627-638 (2015) and D′Uva & Tzahor, “The Key Roles ofERBB2 in Cardiac Regeneration,” Cell Cylce 14(15):2383-2384 (2015),which are hereby incorporated by reference in their entirety).

Yes Associated Protein 1 (YAP) is a transcriptional coactivator, whoseactivation in adult cardiomyocytes has been shown to increasescardiomyocyte proliferation and improve cardiac function aftermyocardial infarction in mice (Lin et al., “Cardiac-Specific YAPActivation Improves Cardiac Function and Survival in an ExperimentalMurine MI Model,” Circ. Res. 115(3):354-363 (2014), which is herebyincorporated by reference in its entirety).

Cyclin D1 is a regulatory subunit of CDK4 and CDK6, whose activity isrequired for cell cycle G1/S transition. Overexpression of cyclin D1results in an increase in CDK4 levels in the adult myocardium, as wellas modest increases in proliferating cell nuclear antigen and CDK2levels (Soonpaa et al., “Cyclin D1 Overexpression Promotes CardiomyocyteDNA Synthesis and Multinucleation in Transgenic Mice,” J. Clin. Invest.99(11):2644-2654 (1997), which is hereby incorporated by reference inits entirety). Expression of cyclin D1 has been shown to promote cellcycle reentry of cardiomyocytes in adult hearts (Lee et al., “CriticalRole of Cyclin D1 Nuclear Import in Cardiomyocyte Proliferation,” Circ.Res. 92(1):e12-19 (2009), which is hereby incorporated by reference inits entirety).

cMYC is highly expressed in fetal, proliferating cardiac myocytes.Although expressed at low levels in the adult heart under normalphysiological conditions, c-Myc expression is rapidly upregulated inresponse to hypertrophic stimuli (Lee et al., “Cell Cycle Re-Entry andMitochondrial Defects in Myc-Mediated Hypertrophic Cardiomyopathy andHeart Failure,” PLoS One 4(9):e7172 (2009), which is hereby incorporatedby reference in its entirety). Activation of cMyc in adult myocardiumhas been shown to provoke cell cycle reentry in post-mitotic mycotyes(Xiao et al., “Inducible Activation of c-Myc in Adult Myocardium In VivoProvokes Cardiac Myocyte Hypertrophy and Reactivation of DNA Synthesis,”Circ. Res. 89(12):1122-1129 (2001), which is hereby incorporated byreference in its entirety).

Exemplary nucleotide sequences encoding suitable cell cycle inducer areshown in Table 2 below.

TABLE 2 Suitable Cell Cycle Inducer Sequences Cell Cycle SEQ InducerNucleotide Sequence ID NO: Lin28ATGGGCTCGGTGTCCAACCAGCAGTTTGCAGGTGGCTGCGCCAAGGCAGCGGAGAAGGC 36 (Mouse)GCCAGAGGAGGCGCCGCCTGACGCGGCCCGAGCGGCAGACGAGCCGCAGCTGCTGCACGGGGCCGGCATCTGTAAGTGGTTCAACGTGCGCATGGGGTTCGGCTTCCTGTCTATGACCGCCCGCGCTGGGGTCGCGCTCGACCCCCCGGTGGACGTCTTTGTGCACCAGAGCAAGCTGCACATGGAAGGGTTCCGAAGCCTCAAGGAGGGTGAGGCGGTGGAGTTCACCTTTAAGAAGTCTGCCAAGGGTCTGGAATCCATCCGTGTCACTGGCCCTGGTGGTGTGTTCTGTATTGGGAGTGAGCGGCGGCCAAAAGGGAAGAACATGCAGAAGCGAAGATCCAAAGGAGACAGGTGCTACAACTGCGGTGGGCTAGACCATCATGCCAAGGAATGCAAGCTGCCACCCCAGCCCAAGAAGTGCCACTTTTGCCAAAGCATCAACCATATGGTGGCCTCGTGTCCACTGAAGGCCCAGCAGGGCCCCAGTTCTCAGGGAAAGCCTGCCTACTTCCGGGAGGAAGAGGAAGAGATCCACAGCCCTGCCCTGCTCCCAGAAGCCCAGAATTGA Pkm2ATGCCGAAGCCACACAGTGAAGCAGGGACTGCCTTCATTCAGACCCAGCAGCTCCATGC 37 (Mouse)AGCCATGGCTGACACCTTCCTGGAACACATGTGCCGCCTGGACATTGACTCTGCCCCCATCACGGCCCGCAACACTGGCATCATTTGTACCATTGGGCCTGCTTCCCGATCTGTGGAGATGCTGAAGGAGATGATTAAGTCTGGAATGAATGTGGCTCGGCTGAATTTCTCTCATGGAACCCATGAGTACCATGCAGAGACCATCAAGAATGTCCGTGAAGCCACAGAAAGCTTTGCATCTGATCCCATTCTCTACCGTCCTGTTGCGGTGGCTCTGGATACAAAGGGACCTGAGATCCGGACTGGACTCATCAAGGGCAGCGGCACCGCTGAGGTGGAGCTGAAGAAGGGAGCCACTCTGAAGATCACCCTGGACAACGCTTACATGGAGAAGTGTGACGAGAACATCCTGTGGCTGGACTACAAGAACATCTGCAAGGTGGTGGAGGTGGGCAGCAAGATCTACGTGGACGATGGGCTCATCTCACTGCAGGTGAAGGAGAAAGGCGCTGACTTCCTGGTGACGGAGGTGGAGAATGGTGGCTCCTTGGGCAGCAAGAAGGGCGTGAACCTGCCGGGCGCTGCTGTGGATCTCCCCGCTGTGTCGGAAAAGGACATCCAGGACCTGAAGTTTGGGGTGGAGCAGGATGTGGACATGGTGTTTGCATCTTTCATCCGCAAGGCAGCCGACGTGCATGAAGTCAGGAAGGTGCTGGGAGAGAAGGGCAAGAACATCAAGATCATCAGCAAAATCGAGAACCATGAAGGCGTCCGCAGGTTTGATGAGATCTTGGAGGCCAGTGATGGGATCATGGTGGCTCGTGGTGACCTGGGCATTGAGATTCCTGCAGAGAAGGTCTTCCTGGCTCAGAAGATGATGATCGGGCGATGCAACCGAGCTGGGAAGCCTGTCATCTGTGCCACACAGATGCTGGAGAGCATGATCAAGAAGCCACGCCCCACCCGTGCTGAAGGCAGTGATGTGGCCAATGCAGTCCTGGATGGAGCAGACTGCATCATGCTGTCTGGAGAAACAGCCAAGGGGGACTACCCTCTGGAGGCTGTTCGCATGCAGCACCTGATTGCCCGAGAGGCAGAGGCTGCCATCTACCACTTGCAGCTATTCGAGGAACTCCGCCGCCTGGCGCCCATTACCAGCGACCCCACAGAAGCTGCCGCCGTGGGTGCCGTGGAGGCCTCCTTCAAGTGCTGCAGTGGGGCCATTATCGTGCTCACCAAGTCTGGCAGGAGTGCTCACCAAGTGGCCAGGTACCGCCCTCGGGCTCCTATCATTGCCGTGACTCGAAATCCCCAGACTGCTCGCCAGGCCCATCTGTACCGTGGCATCTTCCCTGTGCTGTGTAAGGATGCCGTGCTGAATGCCTGGGCTGAGGATGTCGACCTTCGTGTAAACTTGGCCATGGATGTTGGCAAGGCCCGAGGCTTCTTCAAGAAGGGAGATGTGGTCATTGTGCTGACCGGGTGGCGCCCTGGCTCTGGATTCACCAACACCATGCGTGTAGTGCCTGTACCT TGAβ-catenin ATGGCTACTCAAGCTGACCTGATGGAGTTGGACATGGCCATGGAGCCGGACAGAAAAGC 38(Mouse) TGCTGTCAGCCACTGGCAGCAGCAGTCTTACTTGGATTCTGGAATCCATTCTGGTGCCACCACCACAGCTCCTTCCCTGAGTGGCAAGGGCAACCCTGAGGAAGAAGATGTTGACACCTCCCAAGTCCTTTATGAATGGGAGCAAGGCTTTTCCCAGTCCTTCACGCAAGAGCAAGTAGCTGATATTGACGGGCAGTATGCAATGACTAGGGCTCAGAGGGTCCGAGCTGCCATGTTCCCTGAGACGCTAGATGAGGGCATGCAGATCCCATCCACGCAGTTTGACGCTGCTCATCCCACTAATGTCCAGCGCTTGGCTGAACCATCACAGATGTTGAAACATGCAGTTGTCAATTTGATTAACTATCAGGATGACGCGGAACTTGCCACACGTGCAATTCCTGAGCTGACAAAACTGCTAAACGATGAGGACCAGGTGGTAGTTAATAAAGCTGCTGTTATGGTCCATCAGCTTTCCAAAAAGGAAGCTTCCAGACATGCCATCATGCGCTCCCCTCAGATGGTGTCTGCCATTGTACGCACCATGCAGAATACAAATGATGTAGAGACAGCTCGTTGTACTGCTGGGACTCTGCACAACCTTTCTCACCACCGCGAGGGCTTGCTGGCCATCTTTAAGTCTGGTGGCATCCCAGCGCTGGTGAAAATGCTTGGGTCACCAGTGGATTCTGTACTGTTCTACGCCATCACGACACTGCATAATCTCCTGCTCCATCAGGAAGGAGCTAAAATGGCAGTGCGCCTAGCTGGTGGACTGCAGAAAATGGTTGCTTTGCTCAACAAAACAAACGTGAAATTCTTGGCTATTACAACAGACTGCCTTCAGATCTTAGCTTATGGCAATCAAGAGAGCAAGCTCATCATTCTGGCCAGTGGTGGACCCCAAGCCTTAGTAAACATAATGAGGACCTACACTTATGAGAAGCTTCTGTGGACCACAAGCAGAGTGCTGAAGGTGCTGTCTGTCTGCTCTAGCAACAAGCCGGCCATTGTAGAAGCTGGTGGGATGCAGGCACTGGGGCTTCATCTGACAGACCCAAGTCAGCGACTTGTTCAAAACTGTCTTTGGACTCTCAGAAACCTTTCAGATGCAGCGACTAAGCAGGAAGGGATGGAAGGCCTCCTTGGGACTCTAGTGCAGCTTCTGGGTTCCGATGATATAAATGTGGTCACCTGTGCAGCTGGAATTCTCTCTAACCTCACTTGCAATAATTACAAAAACAAGATGATGGTGTGCCAAGTGGGTGGCATAGAGGCTCTTGTACGCACCGTCCTTCGTGCTGGTGACAGGGAAGACATCACTGAGCCTGCCATCTGTGCTCTTCGTCATCTGACCAGCCGGCATCAGGAAGCCGAGATGGCCCAGAATGCCGTTCGCCTTCATTATGGACTGCCTGTTGTGGTTAAACTCCTGCACCCACCATCCCACTGGCCTCTGATAAAGGCAACTGTTGGATTGATTCGAAACCTTGCCCTTTGCCCAGCAAATCATGCGCCTTTGCGGGAACAGGGTGCTATTCCACGACTAGTTCAGCTGCTTGTACGAGCACATCAGGACACCCAACGGCGCACCTCCATGGGTGGAACGCAGCAGCAGTTTGTGGAGGGCGTGCGCATGGAGGAGATAGTAGAAGGGTGTACTGGAGCTCTCCACATCCTTGCTCGGGACGTTCACAACCGGATTGTAATCCGAGGACTCAATACCATTCCATTGTTTGTGCAGTTGCTTTATTCTCCCATTGAAAATATCCAAAGAGTAGCTGCAGGGGTCCTCTGTGAACTTGCTCAGGACAAGGAGGCTGCAGAGGCCATTGAAGCTGAGGGAGCCACAGCTCCCCTGACAGAGTTACTCCACTCCAGGAATGAAGGCGTGGCAACATACGCAGCTGCTGTCCTATTCCGAATGTCTGAGGACAAGCCACAGGATTACAAGAAGCGGCTTTCAGTCGAGCTGACCAGTTCCCTCTTCAGGACAGAGCCAATGGCTTGGAATGAGACTGCAGATCTTGGACTGGACATTGGTGCCCAGGGAGAAGCCCTTGGATATCGCCAGGATGATCCCAGCTACCGTTCTTTTCACTCTGGTGGATACGGCCAGGATGCCTTGGGGATGGACCCTATGATGGAGCATGAGATGGGTGGCCACCACCCTGGTGCTGACTATCCAGTTGATGGGCTGCCTGATCTGGGACACGCCCAGGACCTCATGGATGGGCTGCCCCCAGGTGATAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAA caERBB2ATGAAGCTGCGGCTGCCCGCCTCTCCTGAGACACACCTGGACATGCTGCGGCACCTGTA 39(Mouse and CCAGGGCTGTCAGGTGGTGCAGGGCAACCTGGAACTGACCTACCTGCCCACCAACGCCAhuman) GCCTGAGCTTTCTGCAGGACATCCAGGAAGTGCAGGGCTACGTCCTGATCGCCCACAACCAGGTCCGACAGGTGCCCCTGCAGAGACTGAGAATCGTGCGGGGCACCCAGCTGTTCGAGGACAATTATGCCCTGGCCGTGCTGGACAACGGCGACCCCCTGAACAATACCACCCCTGTGACAGGCGCCAGCCCTGGCGGACTGAGAGAACTGCAGCTGCGGAGCCTGACCGAGATCCTGAAGGGCGGCGTGCTGATCCAGAGAAACCCCCAGCTGTGCTACCAGGACACCATCCTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCCCTGACCCTGATCGACACCAACAGAAGCAGAGCCTGCCACCCCTGCAGCCCCATGTGCAAGGGCTCTAGATGTTGGGGCGAGAGCAGCGAGGACTGCCAGTCCCTGACCAGAACAGTGTGTGCCGGCGGATGCGCCAGATGCAAGGGCCCTCTGCCTACCGATTGCTGCCACGAGCAGTGTGCCGCTGGCTGTACAGGCCCCAAGCACAGCGATTGCCTGGCCTGCCTGCACTTTAACCACAGCGGCATCTGCGAGCTGCACTGCCCTGCCCTGGTCACCTACAACACCGACACCTTCGAGAGCATGCCCAACCCCGAGGGCAGATACACCTTCGGCGCCAGCTGTGTGACCGCCTGCCCCTACAACTACCTGAGCACCGATGTGGGCAGCTGCACCCTCGTGTGCCCCCTGCACAATCAGGAAGTGACCGCCGAGGACGGCACCCAGAGATGCGAGAAGTGCAGCAAGCCCTGCGCCAGAGTGTGCTACGGCCTGGGCATGGAACACCTGAGAGAAGTGCGGGCCGTGACCAGCGCCAATATCCAGGAATTCGCCGGCTGCAAGAAGATCTTTGGCTCCCTGGCCTTTCTGCCCGAGAGCTTCGATGGCGACCCTGCCTCTAATACCGCCCCTCTGCAGCCAGAGCAGCTCCAGGTGTTCGAGACACTGGAAGAGATCACCGGCTACCTGTACATCAGCGCCTGGCCCGACAGCCTGCCCGATCTGAGCGTGTTCCAGAATCTGCAGGTCATCAGAGGCCGGATCCTGCACAACGGCGCCTACAGCCTGACACTGCAGGGCCTGGGAATCAGCTGGCTGGGCCTGAGATCTCTGAGAGAGCTGGGCAGCGGCCTGGCTCTGATCCACCACAACACCCACCTGTGCTTCGTGCACACCGTGCCCTGGGACCAGCTGTTTAGAAACCCTCACCAGGCACTGCTGCACACCGCCAACAGACCCGAGGATGAGTGTGTGGGCGAAGGCCTGGCTTGCCATCAGCTGTGCGCTAGAGGCCACTGTTGGGGCCCTGGACCTACCCAGTGCGTGAACTGCTCCCAGTTCCTGCGGGGCCAGGAATGCGTGGAAGAGTGCAGAGTGCTGCAGGGACTGCCCCGCGAGTACGTGAACGCCAGACACTGCCTGCCTTGCCACCCTGAGTGCCAGCCTCAGAATGGCAGCGTGACCTGCTTCGGCCCTGAGGCCGATCAGTGTGTGGCCTGCGCCCACTACAAGGACCCCCCATTCTGCGTGGCCAGATGCCCTAGCGGCGTGAAGCCCGACCTGAGCTACATGCCCATCTGGAAGTTCCCCGACGAGGAAGGCGCCTGCCAGCCTTGTCCCATCAACTGCACCCACAGCTGCGTGGACCTGGACGACAAGGGCTGTCCTGCCGAGCAGAGAGCCAGCCCCCTGACCTCTATCATCTCCGCCGTGGAAGGCATCCTGCTGGTGGTGGTGCTGGGCGTGGTGTTCGGCATCCTGATCAAGCGGCGGCAGCAGAAGATCCGGAAGTACACCATGCGGCGGCTGCTGCAGGAAACCGAGCTGGTCGAGCCTCTGACACCAAGCGGCGCCATGCCTAACCAGGCCCAGATGCGGATCCTGAAAGAGACAGAGCTGCGGAAAGTGAAGGTGCTGGGATCCGGCGCCTTCGGCACAGTGTACAAGGGAATCTGGATCCCCGACGGCGAGAACGTGAAGATCCCCGTGGCCATCAAGGTGCTGAGAGAGAACACCAGCCCCAAGGCCAACAAAGAGATCCTGGACGAGGCCTACGTGATGGCCGGCGTGGGCAGCCCTTATGTGTCCAGACTGCTGGGCATCTGCCTGACCAGCACCGTGCAGCTGGTCACTCAGCTGATGCCTTACGGCTGCCTGCTGGACCACGTGCGCGAGAATAGAGGCAGACTGGGCAGCCAGGACCTGCTGAACTGGTGCATGCAGATCGCCAAGGGCATGAGCTACCTCGAGGACGTGCGGCTGGTGCACAGAGATCTGGCCGCCAGAAACGTGCTCGTGAAGTCCCCCAACCACGTGAAAATCACCGACTTCGGACTGGCCCGGCTGCTGGACATCGACGAGACAGAGTATCACGCCGACGGCGGCAAGGTGCCCATCAAGTGGATGGCCCTGGAATCCATCCTGCGGCGGAGGTTCACCCACCAGAGCGACGTGTGGTCTTACGGCGTGACCGTGTGGGAGCTGATGACATTCGGAGCCAAGCCCTACGACGGCATCCCCGCCAGAGAGATCCCCGATCTGCTGGAAAAGGGCGAGAGACTGCCCCAGCCCCCCATCTGCACCATCGACGTGTACATGATTATGGTCAAGTGCTGGATGATCGACAGCGAGTGCCGGCCCAGATTCCGCGAGCTGGTGTCCGAGTTCTCCAGAATGGCCCGGGACCCCCAGAGATTCGTGGTCATCCAGAACGAGGACCTGGGCCCTGCCTCCCCCCTGGACTCCACCTTTTACCGGTCCCTGCTGGAAGATGACGACATGGGCGACCTGGTGGACGCCGAGGAATACCTGGTGCCCCAGCAGGGCTTCTTCTGCCCTGATCCTGCTCCTGGCGCTGGCGGCATGGTGCATCACAGACACAGAAGCTCCAGCACCAGAAGCGGAGGCGGCGATCTGACCCTGGGACTGGAACCTTCTGAGGAAGAGGCCCCTAGAAGCCCCCTGGCCCCTAGTGAAGGGGCAGGATCTGATGTGTTCGACGGGGACCTGGGAATGGGCGCTGCCAAAGGACTGCAGAGTCTGCCCACCCACGACCCCAGCCCACTGCAGAGGTACAGCGAGGATCCTACCGTGCCTCTGCCCAGCGAGACAGATGGCTACGTGGCCCCTCTGACCTGTAGCCCCCAGCCCGAGTATGTGAACCAGCCCGATGTGCGGCCTCAGCCTCCTAGCCCTAGAGAAGGACCTCTGCCTGCCGCTAGACCTGCCGGCGCTACCCTGGAAAGACCCAAGACACTGAGCCCCGGCAAGAACGGCGTGGTCAAGGACGTGTTCGCCTTTGGCGGAGCCGTGGAAAACCCCGAGTACCTGACACCTCAGGGCGGAGCAGCACCTCAGCCACACCCTCCACCAGCCTTCAGCCCCGCCTTCGACAACCTGTACTACTGGGATCAGGACCCTCCCGAGAGAGGCGCCCCACCTAGCACCTTTAAGGGCACCCCTACCGCCGAGAATCCTGAGTACCTGGGGCTGGACGTGCCCGTCTAA YAPATGGACTACAAAGACGATGACGACAAGCTTGCGGCCGCGAATTCAAGCTTAGCCACCAT 40 (Mouse)GGACTACAAAGACGATGACGATAAAGCAAGGCTCGAATCGGTACCTAAGGATCCCGGGCAGCAGCCGCCGCCTCAACCGGCCCCCCAGGGCCAAGGGCAGCCGCCTTCGCAGCCCCCGCAGGGGCAGGGCCCGCCGTCCGGACCCGGGCAACCGGCACCCGCGGCGACCCAGGCGGCGCCGCAGGCACCCCCCGCCGGGCATCAGATCGTGCACGTCCGCGGGGACTCGGAGACCGACCTGGAGGCGCTCTTCAACGCCGTCATGAACCCCAAGACGGCCAACGTGCCCCAGACCGTGCCCATGAGGCTCCGGAAGCTGCCCGACTCCTTCTTCAAGCCGCCGGAGCCCAAATCCCACTCCCGACAGGCCAGTACTGATGCAGGCACTGCAGGAGCCCTGACTCCACAGCATGTTCGAGCTCATGCCTCTCCAGCTTCTCTGCAGTTGGGAGCTGTTTCTCCTGGGACACTGACCCCCACTGGAGTAGTCTCTGGCCCAGCAGCTACACCCACAGCTCAGCATCTTCGACAGTCTTCTTTTGAGATACCTGATGATGTACCTCTGCCAGCAGGTTGGGAGATGGCAAAGACATCTTCTGGTCAGAGATACTTCTTAAATCACATCGATCAGACAACAACATGGCAGGACCCCAGGAAGGCCATGCTGTCCCAGATGAACGTCACAGCCCCCACCAGTCCACCAGTGCAGCAGAATATGATGAACTCGGCTTCAGGTCCTCTTCCTGATGGATGGGAACAAGCCATGACTCAGGATGGAGAAATTTACTATATAAACCATAAGAACAAGACCACCTCTTGGCTAGACCCAAGGCTTGACCCTCGTTTTGCCATGAACCAGAGAATCAGTCAGAGTGCTCCAGTGAAACAGCCACCACCCCTGGCTCCCCAGAGCCCACAGGGAGGCGTCATGGGTGGCAGCAACTCCAACCAGCAGCAACAGATGCGACTGCAGCAACTGCAGATGGAGAAGGAGAGGCTGCGGCTGAAACAGCAAGAACTGCTTCGGCAGGAGTTAGCCCTGCGTAGCCAGTTACCAACACTGGAGCAGGATGGTGGGACTCAAAATCCAGTGTCTTCTCCCGGGATGTCTCAGGAATTGAGAACAATGACGACCAATAGCTCAGATCCTTTCCTTAACAGTGGCACCTATCACTCTCGAGATGAGAGTACAGACAGTGGACTAAGCATGAGCAGCTACAGTGTCCCTCGAACCCCAGATGACTTCCTGAACAGTGTGGATGAGATGGATACAGGTGATACTATCAACCAAAGCACCCTGCCCTCACAGCAGAACCGTTTCCCAGACTACCTTGAAGCCATTCCTGGGACAAATGTGGACCTTGGAACACTGGAAGGAGATGGAATGAACATAGAAGGAGAGGAGCTGATGCCAAGTCTGCAGGAAGCTTTGAGTTCTGACATCCTTAATGACATGGAGTCTGTTTTGGCTGCCACCAAGCTAGATAAAGAAAGCTTTCTTACATGGTTATAG Cyclin D1ATGGAACACCAGCTCCTGTGCTGCGAAGTGGAGACCATCCGCCGCGCGTACCCTGACAC 41 (Mouse)CAATCTCCTCAACGACCGGGTGCTGCGAGCCATGCTCAAGACGGAGGAGACCTGTGCGCCCTCCGTATCTTACTTCAAGTGCGTGCAGAAGGAGATTGTGCCATCCATGCGGAAAATCGTGGCCACCTGGATGCTGGAGGTCTGTGAGGAGCAGAAGTGCGAAGAGGAGGTCTTCCCGCTGGCCATGAACTACCTGGACCGCTTCCTGTCCCTGGAGCCCTTGAAGAAGAGCCGCCTGCAGCTGCTGGGGGCCACCTGCATGTTCGTGGCCTCTAAGATGAAGGAGACCATTCCCTTGACTGCCGAGAAGTTGTGCATCTACACTGACAACTCTATCCGGCCCGAGGAGCTGCTGCAAATGGAACTGCTTCTGGTGAACAAGCTCAAGTGGAACCTGGCCGCCATGACTCCCCACGATTTCATCGAACACTTCCTCTCCAAAATGCCAGAGGCGGATGAGAACAAGCAGACCATCCGCAAGCATGCACAGACCTTTGTGGCCCTCTGTGCCACAGATGTGAAGTTCATTTCCAACCCACCCTCCATGGTAGCTGCTGGGAGCGTGGTGGCTGCGATGCAAGGCCTGAACCTGGGCAGCCCCAACAACTTCCTCTCCTGCTACCGCACAACGCACTTTCTTTCCAGAGTCATCAAGTGTGACCCGGACTGCCTCCGTGCCTGCCAGGAACAGATTGAAGCCCTTCTGGAGTCAAGCCTGCGCCAGGCCCAGCAGAACGTCGACCCCAAGGCCACTGAGGAGGAGGGGGAAGTGGAGGAAGAGGCTGGTCTGGCCTGCACGCCCACCGACGTGCGAGATGTGGACATC TGA C-MycATGCCGCTGAACGTGAGCTTTACCAACCGCAACTATGATCTGGATTATGATAGCGTGCA 42 (Mouse)GCCGTATTTTTATTGCGATGAAGAAGAAAACTTTTATCAGCAGCAGCAGCAGAGCGAACTGCAGCCGCCGGCGCCGAGCGAAGATATTTGGAAAAAATTTGAACTGCTGCCGACCCCGCCGCTGAGCCCGAGCCGCCGCAGCGGCCTGTGCAGCCCGAGCTATGTGGCGGTGACCCCGTTTAGCCTGCGCGGCGATAACGATGGCGGCGGCGGCAGCTTTAGCACCGCGGATCAGCTGGAAATGGTGACCGAACTGCTGGGCGGCGATATGGTGAACCAGAGCTTTATTTGCGATCCGGATGATGAAACCTTTATTAAAAACATTATTATTCAGGATTGCATGTGGAGCGGCTTTAGCGCGGCGGCGAAACTGGTGAGCGAAAAACTGGCGAGCTATCAGGCGGCGCGCAAAGATAGCGGCAGCCCGAACCCGGCGCGCGGCCATAGCGTGTGCAGCACCAGCAGCCTGTATCTGCAGGATCTGAGCGCGGCGGCGAGCGAATGCATTGATCCGAGCGTGGTGTTTCCGTATCCGCTGAACGATAGCAGCAGCCCGAAAAGCTGCGCGAGCCAGGATAGCAGCGCGTTTAGCCCGAGCAGCGATAGCCTGCTGAGCAGCACCGAAAGCAGCCCGCAGGGCAGCCCGGAACCGCTGGTGCTGCATGAAGAAACCCCGCCGACCACCAGCAGCGATAGCGAAGAAGAACAGGAAGATGAAGAAGAAATTGATGTGGTGAGCGTGGAAAAACGCCAGGCGCCGGGCAAACGCAGCGAAAGCGGCAGCCCGAGCGCGGGCGGCCATAGCAAACCGCCGCATAGCCCGCTGGTGCTGAAACGCTGCCATGTGAGCACCCATCAGCATAACTATGCGGCGCCGCCGAGCACCCGCAAAGATTATCCGGCGGCGAAACGCGTGAAACTGGATAGCGTGCGCGTGCTGCGCCAGATTAGCAACAACCGCAAATGCACCAGCCCGCGCAGCAGCGATACCGAAGAAAACGTGAAACGCCGCACCCATAACGTGCTGGAACGCCAGCGCCGCAACGAACTGAAACGCAGCTTTTTTGCGCTGCGCGATCAGATTCCGGAACTGGAAAACAACGAAAAAGCGCCGAAAGTGGTGATTCTGAAAAAAGCGACCGCGTATATTCTGAGCGTGCAGGCGGAAGAACAGAAACTGATTAGCGAAGAAGATCTGCTGCGCAAACGCCGCGAACAGCTGAAACATAAACTGGAACAGCTGCGCAACAGCTGCGCGTAA Lin28ATGGGCTCCGTGTCCAACCAGCAGTTTGCAGGTGGCTGCGCCAAGGCGGCAGAAGAGGC 43 (Human)GCCCGAGGAGGCGCCGGAGGACGCGGCCCGGGCGGCGGACGAGCCTCAGCTGCTGCACGGTGCGGGCATCTGTAAGTGGTTCAACGTGCGCATGGGGTTCGGCTTCCTGTCCATGACCGCCCGCGCCGGGGTCGCGCTCGACCCCCCAGTGGATGTCTTTGTGCACCAGAGTAAGCTGCACATGGAAGGGTTCCGGAGCTTGAAGGAGGGTGAGGCAGTGGAGTTCACCTTTAAGAAGTCAGCCAAGGGTCTGGAATCCATCCGTGTCACCGGACCTGGTGGAGTATTCTGTATTGGGAGTGAGAGGCGGCCAAAAGGAAAGAGCATGCAGAAGCGCAGATCAAAAGGAGACAGGTGCTACAACTGTGGAGGTCTAGATCATCATGCCAAGGAATGCAAGCTGCCACCCCAGCCCAAGAAGTGCCACTTCTGCCAGAGCATCAGCCATATGGTAGCCTCATGTCCGCTGAAGGCCCAGCAGGGCCCTAGTGCACAGGGAAAGCCAACCTACTTTCGAGAGGAAGAAGAAGAAATCCACAGCCCTACCCTGCTCCCGGAGGCACAGAATTGA Pkm2ATGCAGTGGAGCTCAGAGAGAGGAGAACGGCTCCTCACGCCTGGGGCCTGCTCTTCAGA 44 (Human)AGTCCCCAGCGCCGTTCCTTCCAGATCAGGCGGCTCTCCAGGGCACACCGTATTCAGCTCTGAGCGGTCTTTGCTAGTGAGGCCAAGGAGCCACCCTGAGCCAAAAGGGGAGCATTATGTCACCGGAAGCCCAACCCCAGAGAACCAAAGGACCTCAGCAGCCATGTCGAAGCCCCATAGTGAAGCCGGGACTGCCTTCATTCAGACCCAGCAGCTGCACGCAGCCATGGCTGACACATTCCTGGAGCACATGTGCCGCCTGGACATTGATTCACCACCCATCACAGCCCGGAACACTGGCATCATCTGTACCATTGGCCCAGCTTCCCGATCAGTGGAGACGTTGAAGGAGATGATTAAGTCTGGAATGAATGTGGCTCGTCTGAACTTGTCTCATGGAACTCATGAGTACCATGCGGAGACCATCAAGAATGTGCGCACAGCCACGGAAAGCTTTGCTTCTGACCCCATCCTCTACCGGCCCGTTGCTGTGGCTCTAGACACTAAAGGACCTGAGATCCGAACTGGGCTCATCAAGGGCAGCGGCACTGCAGAGGTGGAGCTGAAGAAGGGAGCCACTCTCAAAATCACGCTGGATAACGCCTACATGGAAAAGTGTGACGAGAACATCCTGTGGCTGGACTACAAGAACATCTGCAAGGTGGTGGAAGTGGGCAGCAAGATCTACGTGGATGATGGGCTTATTTCTCTCCAGGTGAAGCAGAAAGGTGCCGACTTCCTGGTGACGGAGGTGGAAAATGGTGGCTCCTTGGGCAGCAAGAAGGGTGTGAACCTTCCTGGGGCTGCTGTGGACTTGCCTGCTGTGTCGGAGAAGGACATCCAGGATCTGAAGTTTGGGGTCGAGCAGGATGTTGATATGGTGTTTGCGTCATTCATCCGCAAGGCATCTGATGTCCATGAAGTTAGGAAGGTCCTGGGAGAGAAGGGAAAGAACATCAAGATTATCAGCAAAATCGAGAATCATGAGGGGGTTCGGAGGTTTGATGAAATCCTGGAGGCCAGTGATGGGATCATGGTGGCTCGTGGTGATCTAGGCATTGAGATTCCTGCAGAGAAGGTCTTCCTTGCTCAGAAGATGATGATTGGACGGTGCAACCGAGCTGGGAAGCCTGTCATCTGTGCTACTCAGATGCTGGAGAGCATGATCAAGAAGCCCCGCCCCACTCGGGCTGAAGGCAGTGATGTGGCCAATGCAGTCCTGGATGGAGCCGACTGCATCATGCTGTCTGGAGAAACAGCCAAAGGGGACTATCCTCTGGAGGCTGTGCGCATGCAGCACCTGATAGCTCGTGAGGCTGAGGCAGCCATGTTCCACCGCAAGCTGTTTGAAGAACTTGTGCGAGCCTCAAGTCACTCCACAGACCTCATGGAAGCCATGGCCATGGGCAGCGTGGAGGCTTCTTATAAGTGTTTAGCAGCAGCTTTGATAGTTCTGACGGAGTCTGGCAGGTCTGCTCACCAGGTGGCCAGATACCGCCCACGTGCCCCCATCATTGCTGTGACCCGGAATCCCCAGACAGCTCGTCAGGCCCACCTGTACCGTGGCATCTTCCCTGTGCTGTGCAAGGACCCAGTCCAGGAGGCCTGGGCTGAGGACGTGGACCTCCGGGTGAACTTTGCCATGAATGTTGGCAAGGCCCGAGGCTTCTTCAAGAAGGGAGATGTGGTCATTGTGCTGACCGGATGGCGCCCTGGCTCCGGCTTCACCAACACCATGCGTGTTGTTCCTGTGCCGTGA β-cateninATGGCTACTCAAGCTGATTTGATGGAGTTGGACATGGCCATGGAACCAGACAGAAAAGC 45 (Human)GGCTGTTAGTCACTGGCAGCAACAGTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACTACCACAGCTCCTTCTCTGAGTGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAGTCCTGTATGAGTGGGAACAGGGATTTTCTCAGTCCTTCACTCAAGAACAAGTAGCTGATATTGATGGACAGTATGCAATGACTCGAGCTCAGAGGGTACGAGCTGCTATGTTCCCTGAGACATTAGATGAGGGCATGCAGATCCCATCTACACAGTTTGATGCTGCTCATCCCACTAATGTCCAGCGTTTGGCTGAACCATCACAGATGCTGAAACATGCAGTTGTAAACTTGATTAACTATCAAGATGATGCAGAACTTGCCACACGTGCAATCCCTGAACTGACAAAACTGCTAAATGACGAGGACCAGGTGGTGGTTAATAAGGCTGCAGTTATGGTCCATCAGCTTTCTAAAAAGGAAGCTTCCAGACACGCTATCATGCGTTCTCCTCAGATGGTGTCTGCTATTGTACGTACCATGCAGAATACAAATGATGTAGAAACAGCTCGTTGTACCGCTGGGACCTTGCATAACCTTTCCCATCATCGTGAGGGCTTACTGGCCATCTTTAAGTCTGGAGGCATTCCTGCCCTGGTGAAAATGCTTGGTTCACCAGTGGATTCTGTGTTGTTTTATGCCATTACAACTCTCCACAACCTTTTATTACATCAAGAAGGAGCTAAAATGGCAGTGCGTTTAGCTGGTGGGCTGCAGAAAATGGTTGCCTTGCTCAACAAAACAAATGTTAAATTCTTGGCTATTACGACAGACTGCCTTCAAATTTTAGCTTATGGCAACCAAGAAAGCAAGCTCATCATACTGGCTAGTGGTGGACCCCAAGCTTTAGTAAATATAATGAGGACCTATACTTACGAAAAACTACTGTGGACCACAAGCAGAGTGCTGAAGGTGCTATCTGTCTGCTCTAGTAATAAGCCGGCTATTGTAGAAGCTGGTGGAATGCAAGCTTTAGGACTTCACCTGACAGATCCAAGTCAACGTCTTGTTCAGAACTGTCTTTGGACTCTCAGGAATCTTTCAGATGCTGCAACTAAACAGGAAGGGATGGAAGGTCTCCTTGGGACTCTTGTTCAGCTTCTGGGTTCAGATGATATAAATGTGGTCACCTGTGCAGCTGGAATTCTTTCTAACCTCACTTGCAATAATTATAAGAACAAGATGATGGTCTGCCAAGTGGGTGGTATAGAGGCTCTTGTGCGTACTGTCCTTCGGGCTGGTGACAGGGAAGACATCACTGAGCCTGCCATCTGTGCTCTTCGTCATCTGACCAGCCGACACCAAGAAGCAGAGATGGCCCAGAATGCAGTTCGCCTTCACTATGGACTACCAGTTGTGGTTAAGCTCTTACACCCACCATCCCACTGGCCTCTGATAAAGGCTACTGTTGGATTGATTCGAAATCTTGCCCTTTGTCCCGCAAATCATGCACCTTTGCGTGAGCAGGGTGCCATTCCACGACTAGTTCAGTTGCTTGTTCGTGCACATCAGGATACCCAGCGCCGTACGTCCATGGGTGGGACACAGCAGCAATTTGTGGAGGGGGTCCGCATGGAAGAAATAGTTGAAGGTTGTACCGGAGCCCTTCACATCCTAGCTCGGGATGTTCACAACCGAATTGTTATCAGAGGACTAAATACCATTCCATTGTTTGTGCAGCTGCTTTATTCTCCCATTGAAAACATCCAAAGAGTAGCTGCAGGGGTCCTCTGTGAACTTGCTCAGGACAAGGAAGCTGCAGAAGCTATTGAAGCTGAGGGAGCCACAGCTCCTCTGACAGAGTTACTTCACTCTAGGAATGAAGGTGTGGCGACATATGCAGCTGCTGTTTTGTTCCGAATGTCTGAGGACAAGCCACAAGATTACAAGAAACGGCTTTCAGTTGAGCTGACCAGCTCTCTCTTCAGAACAGAGCCAATGGCTTGGAATGAGACTGCTGATCTTGGACTTGATATTGGTGCCCAGGGAGAACCCCTTGGATATCGCCAGGATGATCCTAGCTATCGTTCTTTTCACTCTGGTGGATATGGCCAGGATGCCTTGGGTATGGACCCCATGATGGAACATGAGATGGGTGGCCACCACCCTGGTGCTGACTATCCAGTTGATGGGCTGCCAGATCTGGGGCATGCCCAGGACCTCATGGATGGGCTGCCTCCAGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAA YAPATGGATCCCGGGCAGCAGCCGCCGCCTCAACCGGCCCCCCAGGGCCAAGGGCAGCCGCC 46 (Human)TTCGCAGCCCCCGCAGGGGCAGGGCCCGCCGTCCGGACCCGGGCAACCGGCACCCGCGGCGACCCAGGCGGCGCCGCAGGCACCCCCCGCCGGGCATCAGATCGTGCACGTCCGCGGGGACTCGGAGACCGACCTGGAGGCGCTCTTCAACGCCGTCATGAACCCCAAGACGGCCAACGTGCCCCAGACCGTGCCCATGAGGCTCCGGAAGCTGCCCGACTCCTTCTTCAAGCCGCCGGAGCCCAAATCCCACTCCCGACAGGCCAGTACTGATGCAGGCACTGCAGGAGCCCTGACTCCACAGCATGTTCGAGCTCATTCCTCTCCAGCTTCTCTGCAGTTGGGAGCTGTTTCTCCTGGGACACTGACCCCCACTGGAGTAGTCTCTGGCCCAGCAGCTACACCCACAGCTCAGCATCTTCGACAGTCTTCTTTTGAGATACCTGATGATGTACCTCTGCCAGCAGGTTGGGAGATGGCAAAGACATCTTCTGGTCAGAGATACTTCTTAAATCACATCGATCAGACAACAACATGGCAGGACCCCAGGAAGGCCATGCTGTCCCAGATGAACGTCACAGCCCCCACCAGTCCACCAGTGCAGCAGAATATGATGAACTCGGCTTCAGCCATGAACCAGAGAATCAGTCAGAGTGCTCCAGTGAAACAGCCACCACCCCTGGCTCCCCAGAGCCCACAGGGAGGCGTCATGGGTGGCAGCAACTCCAACCAGCAGCAACAGATGCGACTGCAGCAACTGCAGATGGAGAAGGAGAGGCTGCGGCTGAAACAGCAAGAACTGCTTCGGCAGGCAATGCGGAATATCAATCCCAGCACAGCAAATTCTCCAAAATGTCAGGAGTTAGCCCTGCGTAGCCAGTTACCAACACTGGAGCAGGATGGTGGGACTCAAAATCCAGTGTCTTCTCCCGGGATGTCTCAGGAATTGAGAACAATGACGACCAATAGCTCAGATCCTTTCCTTAACAGTGGCACCTATCACTCTCGAGATGAGAGTACAGACAGTGGACTAAGCATGAGCAGCTACAGTGTCCCTCGAACCCCAGATGACTTCCTGAACAGTGTGGATGAGATGGATACAGGTGATACTATCAACCAAAGCACCCTGCCCTCACAGCAGAACCGTTTCCCAGACTACCTTGAAGCCATTCCTGGGACAAATGTGGACCTTGGAACACTGGAAGGAGATGGAATGAACATAGAAGGAGAGGAGCTGATGCCAAGTCTGCAGGAAGCTTTGAGTTCTGACATCCTTAATGACATGGAGTCTGTTTTGGCTGCCACCAAGCTAGATAAAGAAAGCTTTCTTACATGGTTATAG Cyclin D1ATGGAACACCAGCTCCTGTGCTGCGAAGTGGAAACCATCCGCCGCGCGTACCCCGATGC 47 (Human)CAACCTCCTCAACGACCGGGTGCTGCGGGCCATGCTGAAGGCGGAGGAGACCTGCGCGCCCTCGGTGTCCTACTTCAAATGTGTGCAGAAGGAGGTCCTGCCGTCCATGCGGAAGATCGTCGCCACCTGGATGCTGGAGGTCTGCGAGGAACAGAAGTGCGAGGAGGAGGTCTTCCCGCTGGCCATGAACTACCTGGACCGCTTCCTGTCGCTGGAGCCCGTGAAAAAGAGCCGCCTGCAGCTGCTGGGGGCCACTTGCATGTTCGTGGCCTCTAAGATGAAGGAGACCATCCCCCTGACGGCCGAGAAGCTGTGCATCTACACCGACAACTCCATCCGGCCCGAGGAGCTGCTGCAAATGGAGCTGCTCCTGGTGAACAAGCTCAAGTGGAACCTGGCCGCAATGACCCCGCACGATTTCATTGAACACTTCCTCTCCAAAATGCCAGAGGCGGAGGAGAACAAACAGATCATCCGCAAACACGCGCAGACCTTCGTTGCCCTCTGTGCCACAGATGTGAAGTTCATTTCCAATCCGCCCTCCATGGTGGCAGCGGGGAGCGTGGTGGCCGCAGTGCAAGGCCTGAACCTGAGGAGCCCCAACAACTTCCTGTCCTACTACCGCCTCACACGCTTCCTCTCCAGAGTGATCAAGTGTGACCCAGACTGCCTCCGGGCCTGCCAGGAGCAGATCGAAGCCCTGCTGGAGTCAAGCCTGCGCCAGGCCCAGCAGAACATGGACCCCAAGGCCGCCGAGGAGGAGGAAGAGGAGGAGGAGGAGGTGGACCTGGCTTGCACACCCACCGACGTGCGGGACGTGGACATC TGA c-MycCTGGATTTTTTTCGGGTAGTGGAAAACCAGCCTCCCGCGACGATGCCCCTCAACGTTAG 48 (Human)CTTCACCAACAGGAACTATGACCTCGACTACGACTCGGTGCAGCCGTATTTCTACTGCGACGAGGAGGAGAACTTCTACCAGCAGCAGCAGCAGAGCGAGCTGCAGCCCCCGGCGCCCAGCGAGGATATCTGGAAGAAATTCGAGCTGCTGCCCACCCCGCCCCTGTCCCCTAGCCGCCGCTCCGGGCTCTGCTCGCCCTCCTACGTTGCGGTCACACCCTTCTCCCTTCGGGGAGACAACGACGGCGGTGGCGGGAGCTTCTCCACGGCCGACCAGCTGGAGATGGTGACCGAGCTGCTGGGAGGAGACATGGTGAACCAGAGTTTCATCTGCGACCCGGACGACGAGACCTTCATCAAAAACATCATCATCCAGGACTGTATGTGGAGCGGCTTCTCGGCCGCCGCCAAGCTCGTCTCAGAGAAGCTGGCCTCCTACCAGGCTGCGCGCAAAGACAGCGGCAGCCCGAACCCCGCCCGCGGCCACAGCGTCTGCTCCACCTCCAGCTTGTACCTGCAGGATCTGAGCGCCGCCGCCTCAGAGTGCATCGACCCCTCGGTGGTCTTCCCCTACCCTCTCAACGACAGCAGCTCGCCCAAGTCCTGCGCCTCGCAAGACTCCAGCGCCTTCTCTCCGTCCTCGGATTCTCTGCTCTCCTCGACGGAGTCCTCCCCGCAGGGCAGCCCCGAGCCCCTGGTGCTCCATGAGGAGACACCGCCCACCACCAGCAGCGACTCTGAGGAGGAACAAGAAGATGAGGAAGAAATCGATGTTGTTTCTGTGGAAAAGAGGCAGGCTCCTGGCAAAAGGTCAGAGTCTGGATCACCTTCTGCTGGAGGCCACAGCAAACCTCCTCACAGCCCACTGGTCCTCAAGAGGTGCCACGTCTCCACACATCAGCACAACTACGCAGCGCCTCCCTCCACTCGGAAGGACTATCCTGCTGCCAAGAGGGTCAAGTTGGACAGTGTCAGAGTCCTGAGACAGATCAGCAACAACCGAAAATGCACCAGCCCCAGGTCCTCGGACACCGAGGAGAATGTCAAGAGGCGAACACACAACGTCTTGGAGCGCCAGAGGAGGAACGAGCTAAAACGGAGCTTTTTTGCCCTGCGTGACCAGATCCCGGAGTTGGAAAACAATGAAAAGGCCCCCAAGGTAGTTATCCTTAAAAAAGCCACAGCATACATCCTGTCCGTCCAAGCAGAGGAGCAAAAGCTCATTTCTGAAGAGGACTTGTTGCGGAAACGACGAGAACAGTTGAAACACAAACTTGAACAGCTACGGAACTCTTGTG CGTAA

In some embodiments, the protein of interest is a reporter protein. Thereporter protein may be a fluorescent protein. Suitable fluorescentproteins include, without limitation, green fluorescent proteins (e.g.,GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, MonomericAzami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins(e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), bluefluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv,Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean,CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (mKate,mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2,DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, mRasberry, mStrawberry,Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange,Monomeric Kusabira-Orange, mTangerine, tdTomato), or any other suitablefluorescent protein. In certain embodiments, the reporter protein is afluorescent protein selected from the group consisting of greenfluorescent protein (GFP), enhanced green fluorescent protein (EGFP),and yellow fluorescent protein (YFP).

In some embodiments, the reporter protein is luciferase. As used herein,the term “luciferase” refers to members of a class of enzymes thatcatalyze reactions that result in production of light. Luciferases havebeen identified in and cloned from a variety of organisms includingfireflies, click beetles, sea pansy (Renilla), marine copepods, andbacteria among others. Examples of luciferases that may be used asreporter proteins include, e.g., Renilla (e.g., Renilla reniformis)luciferase, Gaussia (e.g., Gaussia princeps) luciferase), Metridialuciferase, firefly (e.g., Photinus pyrahs luciferase), click beetle(e.g., Pyrearinus termitilluminans) luciferase, deep sea shrimp (e.g.,Oplophorus gracihrostris) luciferase). Luciferase reporter proteinsinclude both naturally occurring proteins and engineered variantsdesigned to have one or more altered properties relative to thenaturally occurring protein, such as increased photostability, increasedpH stability, increased fluorescence or light output, reduced tendencyto dimerize, oligomerize, aggregate or be toxic to cells, an alteredemission spectrum, and/or altered substrate utilization.

Exemplary nucleotide sequences encoding suitable reporter proteins areshown in Table 3 below.

TABLE 3 Suitable Reporter Protein Sequences Marker Domain SEQ NameNucleotide Sequence ID NO: CopGFPAGAGCGACGAGAGCGGCCTGCCCGCCATGGAGATCGAGTGCCGCATCACCGGCACCCTG 49AACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGGGCACCCCCAAGCAGGGCCGCATGACCAACAAGATGAAGAGCACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCTACCCCAGCGGCTACGAGAACCCCTTCCTGCACGCCATCAACAACGGCGGCTACACCAACACCCGCATCGAGAAGTACGAGGACGGCGGCGTGCTGCACGTGAGCTTCAGCTACCGCTACGAGGCCGGCCGCGTGATCGGCGACTTCAAGGTGGTGGGCACCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAAGATCATCCGCAGCAACGCCACCGTGGAGCACCTGCACCCCATGGGCGATAACGTGCTGGTGGGCAGCTTCGCCCGCACCTTCAGCCTGCGCGACGGCGGCTACTACAGCTTCGTGGTGGACAGCCACATGCACTTCAAGAGCGCCATCCACCCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGCCTTCCGCCGCGTGGAGGAGCTGCACAGCAACACCGAGCTGGGCATCGTGGAGTACCAGCACGCCTTCAAGACCCCCATCGCCTTCGCCAGATCCCGCGCTCAGTCGTCCAATTCTGCCGTGGACGGCACCGCCGGACCCGGCTCCACCGGATCTCGC eGFPATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGA 50CGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAG CTGTACAAGYFP ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGA 51CGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAG CTGTACAAGTAAmCherry ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAA 52GGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAG TAALuciferase ATGGCCGATGCTAAGAACATTAAGAAGGGCCCTGCTCCCTTCTACCCTCTGGAGGATGG53 CACCGCTGGCGAGCAGCTGCACAAGGCCATGAAGAGGTATGCCCTGGTGCCTGGCACCATTGCCTTCACCGATGCCCACATTGAGGTGGACATCACCTATGCCGAGTACTTCGAGATGTCTGTGCGCCTGGCCGAGGCCATGAAGAGGTACGGCCTGAACACCAACCACCGCATCGTGGTGTGCTCTGAGAACTCTCTGCAGTTCTTCATGCCAGTGCTGGGCGCCCTGTTCATCGGAGTGGCCGTGGCCCCTGCTAACGACATTTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATTTCTCAGCCTACCGTGGTGTTCGTGTCTAAGAAGGGCCTGCAGAAGATCCTGAACGTGCAGAAGAAGCTGCCTATCATCCAGAAGATCATCATCATGGACTCTAAGACCGACTACCAGGGCTTCCAGAGCATGTACACATTCGTGACATCTCATCTGCCTCCTGGCTTCAACGAGTACGACTTCGTGCCAGAGTCTTTCGACAGGGACAAAACCATTGCCCTGATCATGAACAGCTCTGGGTCTACCGGCCTGCCTAAGGGCGTGGCCCTGCCTCATCGCACCGCCTGTGTGCGCTTCTCTCACGCCCGCGACCCTATTTTCGGCAACCAGATCATCCCCGACACCGCTATTCTGAGCGTGGTGCCATTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTACCTGATTTGCGGCTTTCGGGTGGTGCTGATGTACCGCTTCGAGGAGGAGCTGTTCCTGCGCAGCCTGCAAGACTACAAAATTCAGTCTGCCCTGCTGGTGCCAACCCTGTTCAGCTTCTTCGCTAAGAGCACCCTGATCGACAAGTACGACCTGTCTAACCTGCACGAGATTGCCTCTGGCGGCGCCCCACTGTCTAAGGAGGTGGGCGAAGCCGTGGCCAAGCGCTTTCATCTGCCAGGCATCCGCCAGGGCTACGGCCTGACCGAGACAACCAGCGCCATTCTGATTACCCCAGAGGGCGACGACAAGCCTGGCGCCGTGGGCAAGGTGGTGCCATTCTTCGAGGCCAAGGTGGTGGACCTGGACACCGGCAAGACCCTGGGAGTGAACCAGCGCGGCGAGCTGTGTGTGCGCGGCCCTATGATTATGTCCGGCTACGTGAATAACCCTGAGGCCACAAACGCCCTGATCGACAAGGACGGCTGGCTGCACTCTGGCGACATTGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGCCTGAAGTCTCTGATCAAGTACAAGGGCTACCAGGTGGCCCCAGCCGAGCTGGAGTCTATCCTGCTGCAGCACCCTAACATTTTCGACGCCGGAGTGGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCTGCCGCCGTCGTCGTGCTGGAACACGGCAAGACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTGACAACCGCCAAGAAGCTGCGCGGCGGAGTGGTGTTCGTGGACGAGGTGCCCAAGGGCCTGACCGGCAAGCTGGACGCCCGCAAGATCCGCGAGATCCTGATCAAGGCTAAGAAAGGCGGCAAGATCGCCGTGTA A

As used herein, the term “transfection” refers to the process by which anucleic acid is introduced into a cell such that they are located insidethe cell. Transfection may refer to the uptake of an exogenous nucleicacid, such as modRNA, mRNA, or a plasmid, by a host cell. For example,modRNA can be used to transfect various cell types (e.g.,cardiomyocytes) with high efficiency, leading to immediate and highlevels of protein expression in a transient, pulse like kinetic(duration of 3-5 days in vitro and 7-10 days in vivo) (see, e.g.,Sultana et al., “Optimizing Cardiac Delivery of Modified mRNA,” Mol.Ther. 25(6):1306-1315 (2017) and Gam et al, “VEGF-A in Patients withType 2 Diabetes,” Nat. Comm. 10:871 (2019), which are herebyincorporated by reference in their entirety).

It has recently been shown that modified mRNA (modRNA) can drive atransient, safe gene expression in the heart with high transfectionlevels without eliciting immune response or compromising the genome(Major & Poss, “Zebrafish Heart Regeneration as a Model for CardiacTissue Repair,” Drug Discov. Today Dis. Models 4(4):219-225 (2007) andHeo & Lee, β-Catenin Mediates Cyclic Strain-Stimulated Cardiomyogenesisin Mouse Embryonic Stem Cells Through ROS-Dependent andIntegrin-Mediated PI3K/Akt Pathways,” J. Cell. Biochem. 112(7):1880-1889(2011), which are hereby incorporated by reference in their entirety).As described in more detail above, modRNA is synthesized by substitutingribonucleotides with modified ribonucleotides. The use of these modifiedribonucleotides results in changing the secondary structure of thesynthesized mRNA, which prevents the Toll-like receptors fromrecognizing the modRNA and permits its translation to a functionalprotein by the ribosomal machinery within the cell, without elicitingimmune response or compromising the genome (Major & Poss, “ZebrafishHeart Regeneration as a Model for Cardiac Tissue Repair,” Drug Discov.Today Dis. Models 4(4):219-225 (2007) and Heo & Lee, β-Catenin MediatesCyclic Strain-Stimulated Cardiomyogenesis in Mouse Embryonic Stem CellsThrough ROS-Dependent and Integrin-Mediated PI3K/Akt Pathways,” J. Cell.Biochem. 112(7):1880-1889 (2011), which are hereby incorporated byreference in their entirety).

According to some embodiments, the first nucleic acid sequence iscapable of increasing translation of a protein of interest in a targetcell relative to when the second nucleic acid sequence encoding theprotein of interest is operatively coupled to a homologous 5′ UTR. Insome embodiments, translation of a protein of interest in the targetcell is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, or more.

As described herein, a target cell may be a mammalian cell. For example,the target cell may be a rodent cell (i.e., mouse or rat cell), rabbitcell, guinea pig cell, feline cell, canine cell, porcine cell, equinecell, bovine cell, ovine cell, monkey cell, or human cell. In certainembodiments, the target cell is a human cell.

Suitable target cells include primary or immortalized cells, at anystage of their lineage, e.g., differentiated cells. Suitabledifferentiated cells include, without limitation, adipocytes,chondrocytes, endothelial cells, epithelial cells (keratinocytes,melanocytes), bone cells (osteoblasts, osteoclasts), liver cells(cholangiocytes, hepatocytes), muscle cells (cardiomyocytes, skeletalmuscle cells, smooth muscle cells), retinal cells (ganglion cells,muller cells, photoreceptor cells), retinal pigment epithelial cells,renal cells (podocytes, proximal tubule cells, collecting duct cells,distal tubule cells), adrenal cells (cortical adrenal cells, medullaryadrenal cells), pancreatic cells (alpha cells, beta cells, delta cells,epsilon cells, pancreatic polypeptide producing cells, exocrine cells),lung cells, bone marrow cells (early B-cell development, early T-celldevelopment, macrophages, monocytes), urothelial cells, fibroblasts,parathyroid cells, thyroid cells, hypothalamic cells, pituitary cells,salivary gland cells, ovarian cells, and testicular cells. In someembodiments, the target cell is a cardiomyocyte or hepatocyte.

Another aspect of the disclosure relates to a pharmaceutical compositioncomprising the nucleic acid molecules described herein.

The pharmaceutical composition may further include a transfectionreagent. In some embodiments, the transfection reagent is a positivelycharged transfection reagent. Suitable transfection reagents are wellknown in the art and include, e.g., Lipofectamine® RNAiMAX(Invitrogen™), Lipofectamine® 2000 (Invitrogen™), Lipofectamine® 3000(Invitrogen™), Invivofectamine™ 3.0 (Invitrogen™), Lipofectamine™MessengerMAX™ (Invitrogen™), Lipofectin™ (Invitrogen™), siLentFet™(Bio-Rad), DharmaFECT™ (Dharmacon), HiPerFect (Qiagen), TranslT-X2®(Mirus), jetMESSENGER® (Polyplus), Trans-Hi™, JetPEI® (Polyplus), andViaFect™ (Promega).

The pharmaceutical composition may further comprise a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier that does not cause an allergic reaction or otheruntoward effect in patients to whom it is administered and arecompatible with the other ingredients in the formulation.Pharmaceutically acceptable carriers include, for example,pharmaceutical diluents, excipients, or carriers suitably selected withrespect to the intended form of administration, and consistent withconventional pharmaceutical practices. For example, solidcarriers/diluents include, but are not limited to, a gum, a starch(e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose,mannitol, sucrose, dextrose), a cellulosic material (e.g.,microcrystalline cellulose), an acrylate (e.g., polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the nucleic acid molecule described herein.

The nucleic acid molecule(s) and/or pharmaceutical composition(s)disclosed herein can be formulated according to any availableconventional method. Examples of preferred dosage forms include atablet, a powder, a subtle granule, a granule, a coated tablet, acapsule, a syrup, a troche, an inhalant, a suppository, an injectable,an ointment, an ophthalmic ointment, an eye drop, a nasal drop, an eardrop, a cataplasm, a lotion and the like. In the formulation, generallyused additives such as a diluent, a binder, a disintegrant, a lubricant,a colorant, a flavoring agent, and if necessary, a stabilizer, anemulsifier, an absorption enhancer, a surfactant, a pH adjuster, anantiseptic, an antioxidant, and the like can be used.

In addition, formulating a pharmaceutical composition can be carried outby combining compositions that are generally used as a raw material forpharmaceutical formulation, according to conventional methods. Examplesof these compositions include, for example, (1) an oil such as a soybeanoil, a beef tallow and synthetic glyceride; (2) hydrocarbon such asliquid paraffin, squalane and solid paraffin; (3) ester oil such asoctyldodecyl myristic acid and isopropyl myristic acid; (4) higheralcohol such as cetostearyl alcohol and behenyl alcohol; (5) a siliconresin; (6) a silicon oil; (7) a surfactant such as polyoxyethylene fattyacid ester, sorbitan fatty acid ester, glycerin fatty acid ester,polyoxyethylene sorbitan fatty acid ester, a solid polyoxyethylenecastor oil and polyoxyethylene polyoxypropylene block co-polymer; (8)water soluble macromolecule such as hydroxyethyl cellulose, polyacrylicacid, carboxyvinyl polymer, polyethyleneglycol, polyvinylpyrrolidone andmethylcellulose; (9) lower alcohol such as ethanol and isopropanol; (10)multivalent alcohol such as glycerin, propyleneglycol, dipropyleneglycoland sorbitol; (11) a sugar such as glucose and cane sugar; (12) aninorganic powder such as anhydrous silicic acid, aluminum magnesiumsilicicate, and aluminum silicate; (13) purified water, and the like.

Additives for use in the above formulations may include, for example,(1) lactose, corn starch, sucrose, glucose, mannitol, sorbitol,crystalline cellulose, and silicon dioxide as the diluent; (2) polyvinylalcohol, polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic,tragacanth, gelatine, shellac, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polypropyleneglycol-poly oxyethylene-block co-polymer, meglumine, calcium citrate,dextrin, pectin, and the like as the binder; (3) starch, agar, gelatinepowder, crystalline cellulose, calcium carbonate, sodium bicarbonate,calcium citrate, dextrin, pectic, carboxymethylcellulose/calcium, andthe like as the disintegrant; (4) magnesium stearate, talc,polyethyleneglycol, silica, condensed plant oil, and the like as thelubricant; (5) any colorant whose addition is pharmaceuticallyacceptable is adequate as the colorant; (6) cocoa powder, menthol,aromatizer, peppermint oil, cinnamon powder as the flavoring agent; (7)antioxidants whose addition is pharmaceutically accepted such asascorbic acid or alpha-tophenol.

Another aspect of the present application relates to a method ofexpressing a protein of interest in a target cell. This method involvesproviding the nucleic acid molecule or a pharmaceutical compositiondescribed herein and contacting a target cell with the nucleic acidmolecule or pharmaceutical composition. The nucleic acid molecule istranslated to express the protein of interest in the target cell.

As described above, the target cell may be a mammalian cell. Forexample, the target cell may be a rodent cell (i.e., mouse or rat cell),rabbit cell, guinea pig cell, feline cell, canine cell, porcine cell,equine cell, bovine cell, ovine cell, monkey cell, or human cell. Incertain embodiments, the target cell is a human cell.

Suitable target cells are described in detail above and include, withoutlimitation, adipocytes, chondrocytes, endothelial cells, epithelialcells (keratinocytes, melanocytes), bone cells (osteoblasts,osteoclasts), liver cells (cholangiocytes, hepatocytes), muscle cells(cardiomyocytes, skeletal muscle cells, smooth muscle cells), retinalcells (ganglion cells, muller cells, photoreceptor cells), retinalpigment epithelial cells, renal cells (podocytes, proximal tubule cells,collecting duct cells, distal tubule cells), adrenal cells (corticaladrenal cells, medullary adrenal cells), pancreatic cells (alpha cells,beta cells, delta cells, epsilon cells, pancreatic polypeptide producingcells, exocrine cells); lung cells, bone marrow cells (early B-celldevelopment, early T-cell development, macrophages, monocytes),urothelial cells, fibroblasts, parathyroid cells, thyroid cells,hypothalamic cells, pituitary cells, salivary gland cells, ovariancells, and testicular cells. In a particular embodiment, the target cellis a cardiomyocyte or hepatocyte.

In certain embodiments of the methods described herein, the target cellis an ischemic cell. The term “ischemic” or “ischemia” refers to areduction in blood flow. Ischemia is associated with a reduction innutrients, including oxygen, delivered to tissues. Ischemia may arisedue to conditions such as atherosclerosis; formation of a thrombus in anartery or vein; or blockage of an artery or vein by an embolus, vascularclosure due to other causes, e.g., vascular spasm. Such conditions mayreduce blood flow, producing a state of hypoperfusion to an organ ortissue, or block blood flow completely. Other conditions that canproduce ischemia include tissue damage due to trauma or injury, such as,e.g., spinal cord injury or viral infection, which can lead to, e.g.,congestive heart failure.

The term “ischemic condition” refers to acute ischemic conditions,including myocardial infarction (MI), ischemic stroke, pulmonaryembolism, perinatal hypoxia, circulatory shock (e.g., hemorrhagic,septic, cardiogenic, mountain sickness, acute respiratory failure) andchronic ischemic conditions, including atherosclerosis, chronic venousinsufficiency, chronic heart failure, cardiac cirrhosis, diabetes,macular degeneration, sleep apnea, Raynaud's disease, systemicsclerosis, nonbacterial thrombotic endocarditis, occlusive arterydisease, angina pectoris, TIAs, and chronic alcoholic liver disease.Ischemic conditions may also result when individuals are placed undergeneral anesthesia, which can cause tissue damage in organs prepared fortransplant. Myocardial ischemic conditions (e.g., myocardial infarction)result in damage to cardiomyocytes.

In some embodiments, the target cell is an ischemic cardiomyocyte. Whenthe target cell is a cardiomyocyte, the nucleic acid molecule may betranslated to express the protein of interest in the heart.

Liver ischemia is a major complication in many clinical scenarios, suchas liver resection, liver transplantation, and trauma (see, e.g.,Konishi et al., “Hepatic Ischemia/Reperfusion: Mechanisms of TissueInjury, Repair, and Regeneration,” Gene Expr. 17(4):277-287, which ishereby incorporated by reference in its entirety). Hepatocytes, whichcomprise the main parenchymal tissue of the liver, may be damaged duringliver ischemia. Thus, in some embodiments, the target cell is anischemic hepatocyte. When the target cell is a hepatocyte, the nucleicacid molecule may be translated to express the protein of interest inthe liver.

According to some embodiments, both cardiomyocytes and hepatocytes arecontacted, and the nucleic acid molecule is translated to express theprotein of interest in the heart and the liver.

In some embodiments, the contacting is carried out after an ischemicevent in the target cell. Suitable ischemic events are associated withischemic conditions, which are described in detail above and include,e.g., myocardial infarction (MI), ischemic stroke, pulmonary embolism,perinatal hypoxia, and circulatory shock.

The Examples below demonstrate the ability of a modRNA constructcomprising a first nucleic acid sequence having at least a portion of a5′ UTR of a carboxylase gene and a second nucleic acid sequence encodinga reporter protein to selectively enhance translation of the reporterprotein in cells that have undergone ischemia as compared tonon-ischemic cells. Thus, in some embodiments, the contacting iseffective to increase translation of the protein of interest in anischemic target cell relative to a non-ischemic target cell. Suitableproteins of interest are described in detail above and include, e.g.,Lin28 and Pkm2.

modRNA constructs can be used for the transient expression of targetproteins of interest. Thus, in some embodiments, the protein of interestis transiently expressed. The protein of interest may be transientlyexpressed for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days.In some embodiments, the protein of interest is transiently expressedfor 3-7 days.

A further aspect of the present application relates to a method oftreating a subject for cardiac ischemia or hepatic ischemia. This methodinvolves providing the nucleic acid molecule or a pharmaceuticalcomposition described herein and contacting the subject with the nucleicacid molecule or pharmaceutical composition described herein, where thenucleic acid molecule is translated to express a protein of interest inthe subject's heart or liver to treat the subject for cardiac ischemiaor hepatic ischemia.

In carrying out the methods of the present application, “treating” or“treatment” includes inhibiting, preventing, ameliorating or delayingonset of a particular condition. Treating and treatment also encompassesany improvement in one or more symptoms of the condition or disorder.Treating and treatment encompasses any modification to the condition orcourse of disease progression as compared to the condition or disease inthe absence of therapeutic intervention.

The terms “disorders” and “diseases” are used inclusively and refer toany condition deviating from normal. Thus, the term “ischemiccondition(s)” refers to any condition, disease, or disorder that isassociated with ischemia.

Suitable subjects for treatment according to the methods of the presentapplication include, without limitation, domesticated and undomesticatedanimals such as rodents (mouse or rat), cats, dogs, rabbits, horses,sheep, pigs, and monkeys. In some embodiments the subject is a humansubject. Exemplary human subjects include, without limitation, infants,children, adults, and elderly subjects.

In some embodiments, the subject is in need of treatment for an acuteischemic condition. Suitable acute ischemic conditions are described indetail above and include, e.g., myocardial infarction (MI), ischemicstroke, pulmonary embolism, perinatal hypoxia, and circulatory shock.

In some embodiments, the subject is in need of treatment for a chronicischemic condition. Suitable chronic ischemic conditions are describedabove and include, e.g., atherosclerosis, chronic venous insufficiency,chronic heart failure, cardiac cirrhosis, diabetes, maculardegeneration, sleep apnea, Raynaud's disease, systemic sclerosis,nonbacterial thrombotic endocarditis, occlusive artery disease, anginapectoris, TIAs, and chronic alcoholic liver disease.

In some embodiments, the subject is in need of treatment for a disordercharacterized by insufficient cardiac function. In some embodiments, themethods disclosed herein are useful for treatment of a disease ordisorder which is congestive heart failure, cardiomyopathy, myocardialinfarction, tissue ischemia, cardiac ischemia, vascular disease,acquired heart disease, congenital heart disease, atherosclerosis,cardiomyopathy, dysfunctional conduction systems, dysfunctional coronaryarteries, pulmonary heard hypertension. In some embodiments, the diseaseis selected from the group consisting of congestive heart failure,coronary artery disease, myocardial infarction, myocardial ischemia,atherosclerosis, cardiomyopathy, idiopathic cardiomyopathy, cardiacarrhythmias, muscular dystrophy, muscle mass abnormality, muscledegeneration, infective myocarditis, drug- or toxin-induced muscleabnormalities, hypersensitivity myocarditis, an autoimmune endocarditis,congenital heart disease, and combinations thereof.

In some embodiments, the contacting is effective to reduce at least onesymptom of an ischemic disease or condition that is associated with theloss or dysfunction of the target cell type. In other embodiments, thecontacting is effective to mediate an improvement in the ischemicdisease or condition that is associated with the loss or dysfunction ofthe target cell type. In certain embodiments, the contacting iseffective to prolong survival in the subject following an ischemic eventas compared to expected survival if no contacting were carried out.

The methods described herein may be carried out to treat a subject forcardiac ischemia, hepatic ischemia, or both cardiac and hepaticischemia. In some embodiments, the subject is treated for cardiacischemia and cardiomyocytes in the subject are contacted with thenucleic acid molecule. In other embodiments, the subject is treated forhepatic ischemia and hepatocytes in the subject are contacted with thenucleic acid molecule. When the method is carried out to treat thesubject for both cardiac ischemia and hepatic ischemia, cardiomyocytesand hepatocytes in the subject are contacted with the nucleic acidmolecule.

In some embodiments, cardiomyocytes and hepatocytes in the subject arecontacted with the pharmaceutical composition described herein.According to such embodiments, the contacting is carried out byinjection.

As described herein above, the protein of interest may be transientlyexpressed. The term “transient expression” refers to expression of aprotein of interest from a non-integrated transgene for a period ofhours, days, or weeks, where the period of time of expression is lessthan the period of time for expression of the protein of interest if thetransgene were integrated into the genome or contained within a stableplasmid replicon in a target host cell.

The protein of interest may be a cell cycle inducer. Suitable cell cycleinducer proteins are described above and include, e.g., Lin28 andPyruvate Kinase Muscle Isozyme M2 (Pkm2).

In some embodiments, the contacting is effective to deliver the nucleicacid molecule or pharmaceutical composition described herein to aspecific tissue in the subject. The tissue may be muscle tissue. Forexample, the muscle tissue may be skeletal muscle, cardiac muscle, orsmooth muscle. In some embodiments, the tissue is the myocardium.

Contacting, according to the methods of the present application, may becarried out orally, topically, transdermally, parenterally,subcutaneously, intravenously, intramuscularly, intraperitoneally, byintranasal instillation, by intracavitary or intravesical instillation,intraocularly, intraarterially, intralesionally, or by application tomucous membranes. Thus, in some embodiments, the contacting is carriedout intramuscularly, intravenously, subcutaneously, orally, orintraperitoneally. In specific embodiments, the contacting is carriedout by direct intra-myocardial injection.

Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents.

Suitable regimens for initial contacting and further doses or forsequential contacting steps may all be the same or may be variable.Appropriate regimens can be ascertained by the skilled artisan, fromthis disclosure, the documents cited herein, and the knowledge in theart.

An in vitro dosage unit (e.g., for contacting target cells in a 6-well,12-well, 24-well, or 96-well plate) can include from, for example, 0.1to 10 μg, 0.5 to 10 μg, 1 to 5 μg, 1 to 10 μg, 1 to 15 μg, and 1 to 20μg (e.g., 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8μg, 0.9 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg,11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg) ofa compound described herein.

An in vivo dosage unit (e.g., for contacting target cells within asubject) can include from, for example, 1 to 100 μg, 10 to 100 μg, 15 to100 μg, 20 to 100 μg, 25 to 100 μg, and 1 to 200 μg (e.g., 1 μg, 2 μg, 3μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg) of acompound described herein. In some embodiments, the in vivo dosage unitincludes, for example, 1 to 10 mg, 1 to 20 mg, 1 to 30 mg, 1 to 40 mg, 1to 50 mg, 1 to 60 mg, 1 to 70 mg, 1 to 80 mg, 1 to 90 mg, 1 to 100 mg,10 to 100 mg, 20 to 100 mg, 30 to 100 mg, 40 to 100 mg, 50 to 100 mg, 60to 100 mg, 70 to 100 mg, 80 to 100 mg, and 90 to 100 mg (e.g., 1 mg, 2mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85mg, 90 mg, 95 mg, 100 mg) of a compound described herein.

In some embodiments, a subject is contacted with the nucleic acidmolecule or pharmaceutical composition described herein in one dose. Inother embodiments, the subject is contacted with the nucleic acidmolecule or pharmaceutical composition described herein in a series oftwo or more doses in succession. In some other embodiments, where thesubject is contacted with the nucleic acid molecule or pharmaceuticalcomposition described herein in a single dose, in two doses, and/or morethan two doses, the doses may be the same or different, and they areadministered with equal or with unequal intervals between them.

A subject may be contacted with the nucleic acid molecule orpharmaceutical composition described herein in many frequencies over awide range of times. In some embodiments, the subject is contacted overa period of less than one day. In other embodiments, the subject iscontacted over two, three, four, five, or six days. In some embodiments,the contacting is carried out one or more times per week, over a periodof weeks. In other embodiments, the contacting is carried out over aperiod of weeks for one to several months. In various embodiments, thecontacting is carried out over a period of months. In others, thecontacting may be carried out over a period of one or more years.Generally, lengths of treatment will be proportional to the length ofthe ischemic disease process, the effectiveness of the therapies beingapplied, and the condition and response of the subject being treated.According to some embodiments, the contacting is carried out daily.

The choice of formulation for contacting a subject with the nucleic acidmolecule or pharmaceutical composition described herein will depend on avariety of factors. Prominent among these will be the species ofsubject, the nature of the disorder, dysfunction, or disease beingtreated and its state and distribution in the subject, the nature ofother therapies and agents that are being administered, the optimumroute for administration, survivability via the route, the dosingregimen, and other factors that will be apparent to those skilled in theart. In particular, for instance, the choice of suitable carriers andother additives will depend on the exact route of contacting and thenature of the particular dosage form.

Yet another aspect of the present application relates to a method ofidentifying a 5′ untranslated region (5′ UTR) for selectively enhancingtranslation of a heterologous protein of interest in a target cell ortissue. This method involves obtaining a first sample of living tissuecomprising a target cell under disease conditions and a second sample ofliving tissue comprising the target cell under non-disease conditions;quantifying genes that are transcribed and translated in the first andsecond samples; identifying genes which (i) are transcribed at similaror lower levels in the first sample relative to the second sample and(ii) are translated at higher levels in the first sample relative to thesecond sample; and identifying the 5′ UTR of the identified genes, wherethe identified 5′ UTR is capable of selectively enhancing translation ofa heterologous protein of interest in a target cell or tissue.

FIG. 11 is a flow diagram illustrating one embodiment of a method ofidentifying a 5′ untranslated region (5′ UTR) described herein. In FIG.10A, step A corresponds to the step of obtaining a disease sample (i.e.,a first sample of living tissue comprising a target cell under diseaseconditions) and a reference sample (i.e., second sample of living tissuecomprising the target cell under non-disease conditions); step Bcorresponds to proteome analysis (i.e., quantifying genes that aretranslated) and transcriptome analysis (i.e., quantifying genes that aretranscribed) in the disease and reference samples (i.e., the first andsecond samples); step C corresponds to identifying genes which (i) aretranscribed at similar or lower levels in the first sample relative tothe second sample and (ii) are translated at higher levels in the firstsample relative to the second sample; and step D corresponds toidentifying the 5′ UTR of the identified genes, where the identified 5′UTR is capable of selectively enhancing translation of a heterologousprotein of interest in a target cell or tissue.

In some embodiments, the identified 5′ UTR corresponds to a gene thatencodes a protein that is translated at least 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2.0-fold, 2.1-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold,2.8-fold, 2.9-fold, or 3.0-fold greater under a disease condition thanunder a reference condition. The identified 5′ UTR may correspond to agene that encodes a protein that is transcribed at least 0.9-fold,0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, or0.1-fold less under a disease condition than under a referencecondition.

The method may further involve selecting genes with a 5′ UTR of lessthan 100 nucleotides before or after identifying the 5′ UTR of theidentified genes.

In some embodiments, the method further involves providing a firstmodified mRNA (modRNA) construct encoding a reporter protein operablycoupled to the identified 5′ UTR; providing a second modRNA constructencoding a reporter protein operably coupled to a reference 5′ UTR;expressing the first modRNA construct and the second modRNA construct ina cell or tissue of interest under disease conditions and non-diseaseconditions; measuring the expression of the reporter protein from eachof the first and second modRNA constructs under disease and non-diseaseconditions; and determining whether the identified 5′ UTR of the firstmodRNA construct selectively enhances protein translation in a diseasetissue as compared to the reference 5′ UTR of the second modRNAconstruct.

The method according to this aspect of the present application mayfurther involve providing a modRNA molecule comprising a heterologousprotein of interest operably coupled to a portion of the identified 5′UTR and comparing translation of the protein of interest in the targetcell under disease conditions to the translation of the protein ofinterest in the target cell under non-disease conditions; andidentifying, based on said comparing, a nucleic acid sequence thatselectively enhances translation of the protein of interest from themodRNA molecule under disease conditions. In some embodiments, theprotein of interest is not a reporter protein. The protein of interestmay comprise a cell cycle inducer. Suitable cell cycle inducers aredescribed above and include, e.g., Lin28 and Pkm2.

Suitable modRNA modifications are described in detail above. In someembodiments, the modRNA comprises pseudouridine or methylpseudouridine.

In some embodiments, the disease condition is ischemia. Suitableischemic conditions are described in detail above and include, e.g.,acute and chronic ischemic conditions.

In some embodiments, the disease is cancer, an autoimmune disorder,bacterial infection, viral infection, inflammation, fibrotic disorder,metabolic disorder, a neoplasm, cardiovascular or cerebrovasculardisorder, a skin disorder, or any disease or disorder in which it is ormay be desirable to express a therapeutic protein of interest to improvethe disease condition in a cell, tissue, or subject.

According to certain embodiments, the samples are obtained from amammal. For example, the sample may be a rodent (e.g., a mouse or rat)sample, a rabbit sample, a guinea pig sample, a feline sample, a caninesample, a porcine sample, an equine sample, a bovine sample, an ovinesample, a non-human primate (e.g., a monkey) sample, or human sample. Insome embodiments, the mammal is a rodent or a human. The human may be,without limitation, an infant, a child, an adult, or an elderly adult.

The present application may be further illustrated by reference to thefollowing examples.

EXAMPLES

The examples below are intended to exemplify the practice of embodimentsof the disclosure but are by no means intended to limit the scopethereof.

Materials and Methods for Examples 1-7

Mice

All animal procedures were performed according to protocols approved bythe Icahn School of Medicine at Mount Sinai Institutional Care and UseCommittee. CFW mice were used for the study. Before surgery, mice wereanesthetized with a cocktail of 100 mg/kg ketamine and 10 mg/kgxylazine. For protein expression, mice received 25 μg of modRNA incitrate buffer injected directly into the myocardium during open-chestsurgery. When required, 25 μg of modRNA was injected into the borderzone with three injections immediately after left anterior descendingartery (LAD) ligation.

ModRNA Synthesis

Clean PCR products generated with plasmid templates (GeneArt,ThermoFisher Scientific) were used as the template for mRNA. modRNAswere generated by transcription in vitro with a customizedribonucleoside blend of anti-reverse cap analog, 3′-O-Me-m7G(5′)ppp(5′)G(6 mM, TriLink Biotechnologies) or with Anti-Reverse Cap Analog (ARCA)or CleanCap® Reagent AG, guanosine triphosphate (1.5 mM, LifeTechnologies), adenosine triphosphate (7.5 mM, Life Technologies),cytidine triphosphate (7.5 mM, Life Technologies), andN1-methylpseudouridine-5-triphosphate (7.5 mM, TriLink Biotechnologies).The mRNA was purified with the MEGAclear™ kit (Life Technologies) andtreated with Antarctic Phosphatase (New England Biolabs). It was thenre-purified with the MEGAclear™ kit. The mRNA was quantified on aNanoDrop™ spectrometer (ThermoFisher Scientific), precipitated withethanol and ammonium acetate, and resuspended in 10 mM Tris-HCl and 1 mMEDTA.

Rat Neonatal Cardiomyocytes (RNCM) Isolation

Ventricular RNCMs were isolated from 3-4 day-old Sprague-Dawley rat pupsusing the Pierce™ Primary Cardiomyocyte Isolation Kit (ThermoFisherScientific, Catalog No. 88282). After isolation, cells were incubated in10% horse serum DMEM; after 16 hours, media was changed, cardiomyocytegrowth supplement was added, and cells were transfected with modRNA.

In Vitro modRNA Transfection

Either 2.5 μg/well in a 24-well plate of luciferase (Luc) modRNA or 10μg/well in a 6-well plate of nGFP modRNA was transfected into neonatalrat cardiomyocytes (CMs) using the transfection reagent JetPEI®(Polyplus). The transfection mixture was prepared according to themanufacturer's protocol and then added to cells cultured in DMEM mediumcontaining 10% fetal bovine serum (FBS) and Anti-anti. Then, 24-hourspost-transfection, either cells were imaged to measure expression levelin IVIS® (FIGS. 5A-5F) or cell lysates were collected and analyzed bywestern blot.

Heart Ischemic Injury

Myocardial Infarction (MI) was induced by permanently ligating the leftanterior descending artery (LAD). The left thoracic region was shavedand sterilized. After intubation, the heart was exposed by leftthoracotomy. The LAD was ligated with a suture. Mouse hearts without(sham) or with ischemic injury (MI) were collected at 4 and 23 hourspost-MI (FIG. 1A). The ischemic area tissue (or equivalent area insham-operated hearts) was collected, divided into two pieces, andquickly snap-frozen. Half of the ischemic heart tissues was sent forRNAseq while the other half was sent for proteomics analysis to evaluategene and protein fold change, respectively, between MI versussham-operated hearts.

When required, 25 μg modRNA was injected into the infarct border zoneimmediately after LAD ligation. The thoracotomy and skin were suturedclosed in layers. Excess air was removed from the thoracic cavity, andthe mouse was removed from ventilation when normal breathing wasestablished.

Liver Ischemic Injury

Liver ischemia was induced by closing left lateral lobe and median foran hour. Three injections of 25 μg modRNA were injected into the leftlateral lobe immediately after the clip was removed.

Kidney Ischemic Injury

Kidney ischemia was induced by applying a micro clip to the renal arteryand renal vein. Successful ischemia can be visually confirmed by agradual uniform darkening of the kidney. The clip was removed after 30minutes and three injections of 25 μg modRNA were injected into thekidney.

In Vivo modRNA Delivery

25 μg Luc modRNA in a total volume of 60 μl in TB buffer was deliveredvia direct injection to the myocardium. The sucrose-citrate buffercontained 20 μl sucrose in nuclease-free water (0.3 g/mL) and 20 μlcitrate (0.1 M, pH 7; Sigma) mixed with 20 μL modRNA. The transfectionmixture was directly injected (three individual injections, 20 μl each)into the heart, kidney, or liver.

Detecting of Luciferase Expression Using the IVIS® In Vivo ImagingSystem

Bioluminescence imaging of the transfected cells (24-72 hours) orinjected mice was taken at different time points (4-144 hours) using theIVIS® system. To visualize cells expressing Firefly luciferase (Luc) invitro, D-luciferin was added to cell culture plates and images weretaken in the IVIS® system (IVIS® Spectrum NCRR S10-RR026561-01). Tovisualize cells expressing Renilla luciferase in vitro, cells werewashed twice with media and Renilla luciferase substrate was added tocell culture plates. Images were taken using an emission filter 500. Tovisualize tissues expressing Luc in vivo, mice were anesthetized withisoflurane (Abbott Laboratories), and luciferin (150m/g body weight;Sigma) was injected intraperitoneally. Mice were imaged using the IVIS®imaging system every 2 minutes until the Luc signal reached a plateau.Imaging data were analyzed and quantified with Living Image® software(PerkinElmer).

Western Blotting

Cell lysates were collected and subjected to SDS-PAGE in 12% precastNupage Bis/Tris gels (Invitrogen) under reducing conditions in IVIESrunning buffer (Invitrogen). The resulting bands were transferred onto anitrocellulose membrane (Bio-Rad) by blotting in a semidry transferapparatus with Nupage-MOPS transfer buffer (Invitrogen). The membranewas blocked by incubation with TB S/TWEEN® containing 5% dry milk powderand incubated with specific primary antibodies overnight at 4° C. It wasthen washed in TBS/TWEEN® and incubated with rabbit or goat secondaryantibodies conjugated to horseradish peroxidase for 1 hour at roomtemperature. Antibody binding was detected with an enhancedchemiluminescence (ECL) detection system (Pierce). Prestained proteinstandards (Amersham) were used to determine molecular weight.

RNA Isolation

Total RNA was isolated with RNeasy® Mini Kit (QIAGEN) and DNA wasdegraded by treatment with TURBO DNase (Invitrogen). RNA quality waschecked by bioanalyzer.

RNA Sequencing

Poly(A)-tailed RNA was prepared by the Epigenomics Core at CornellMedical College, with the mRNA Seq Sample Prep Kit (Illumina), and usedto create libraries for HiSeq2000 sequencing (Illumina). Single 50 bpreads were used for sequencing. A mean of 30 million reads per samplewas obtained, with a mean quality score of 35.2. Partek® Flow® softwarewas used for data analysis. RNA-Seq reads were aligned to mm10 with STARversion 2.53a. Read counts were generated by applying the Partek E/Malgorithm to UCSC RefSeq 2017-08-02. Counts were normalized with the TMMalgorithm and the Partek® Flow® GSA algorithm was used for statisticalanalysis. The RNAseq data used in this study are available usingaccession #GSE138201, which is hereby incorporated by reference in itsentirety.

Protein Mass Spectrometry

All solvents were HPLC grade, from Sigma-Aldrich, and all chemicalswhere not stated otherwise were obtained from Sigma-Aldrich. For amplepreparation, samples were lysed in Biognosys' Lysis Buffer with aTissueLyserII bead mill (QIAGEN) using stainless steel grinding beadsfor 3 minutes at 30 Hz. Samples were treated with benzonase after lysisto reduce DNA contamination. Protein concentrations of the lysates wereestimated using a UV/VIS Spectrometer (SPECTROstar Nano, BMG LABTECH).Approximately 100 μg of protein from each sample were denatured usingBiognosys' Denature Buffer, reduced using Biognosys' Reduction Solutionfor 60 minutes at 37° C. and alkylated using Biognosys' AlkylationSolution for 30 minutes at room temperature in the dark. Subsequently,digestion to peptides was carried out using 3 μg of trypsin (Promega)overnight at 37° C.

Peptides were desalted using C18 MacroSpincolumns (The Nest Group)according to the manufacturer's instructions and dried down using aSpeedVac system. Next, peptides were resuspended in 50 μl LC solvent A(1% acetonitrile, 0.1% formic acid (FA)) and spiked with Biognosys' iRTkit calibration peptides. Peptide concentrations were determined using aUV/VIS Spectrometer (SPECTROstar Nano, BMG LABTECH).

For HPRP fractionation, equal-volume samples from each condition werepooled. Ammonium hydroxide was added to all pools to a pH value>10. Thefractionation was performed using a Dionex UltiMate 3000 RS Pump (ThermoScientific) on an Acquity UPLC CSH C18 1.7 μm, 2.1×150 mm column(Waters). The gradient was 2% to 35% Solvent B in 10 min; solvents wereA: 20 mM ammonium formate in H₂O, B: Acetonitrile. Fractions were takenevery 15 sec and sequentially pooled to 4 fraction pools. These weredried down and resolved in 20 μl solvent A. Prior to mass spectrometricanalyses, they were spiked with Biognosys' iRTkit calibration peptides.Peptide concentrations were determined using a UV/VIS Spectrometer(SPECTROstar Nano, BMG LABTECH).

For the LC-MS/MS (shotgun) measurements, 2 μg of peptides were injectedto an in-house packed C18 column (ReproSil-Pur® (Maisch), 1.9 μmparticle size, 120 Å pore size; 75 μm inner diameter, 50 cm length, NewObjective) on a nano-liquid chromatography system (Easy nLC™ 1200System, ThermoFisher Scientific) connected to a Q Exactive™ HF(ThermoFisher Scientific) mass spectrometer equipped with a standardnano-electrospray source. LC solvents were A: 1% acetonitrile in waterwith 0.1% FA; B: 15% water in acetonitrile with 0.1% FA. The non-linearLC gradient was 1-55% solvent B in 60 minutes followed by 55-90% B in 10seconds, 90% B for 10 minutes, 90%-1% B for 10 seconds and 1% B for 5minutes. A modified TOP15 method was used (Scheltema et al., “The QExactive HF, a Benchtop Mass Spectrometer with a Pre-Filter,High-Performance Quadrupole and an Ultra-High-Field Orbitrap Analyzer,”Mol. Cell Proteomics 13:3698-3708 (2014), which is hereby incorporatedby reference in its entirety).

The mass spectrometric data were analyzed using Biognosys' search enginePulsar (version 1.0.16105), with the false discovery rate on peptide andprotein level set to 1%. A mouse UniProt FASTA database (Mus musculus,2017-07-01) was used for the search engine, allowing for 2 missedcleavages and variable modifications (N-term acetylation, methionineoxidation).

For the LC-MS/MS HRM measurements, 2 μg of peptides per sample wereinjected into C18 column (ReproSil-Pur® (Maisch), 1.9 μm particle size,120 Å pore size; 75 μm inner diameter, 50 cm length, New Objective) on anano-liquid chromatography system (Easy nLC™ 1200 System, ThermoFisherScientific) connected to a Q Exactive™ HF (ThermoFisher Scientific) massspectrometer equipped with a standard nano-electrospray source. LCsolvents were A: 1% acetonitrile in water with 0.1% formic acid; B: 15%water in acetonitrile with 0.1% formic acid. The nonlinear LC gradientwas 1-52% solvent B in 60 minutes followed by 52-90% B in 10 seconds and90% B for 10 minutes. A DIA method with one full range survey scan and14 DIA windows was used.

HRM mass spectrometric data were analyzed using Spectronaut™ Pulsarsoftware (Biognosys). The false discovery rate on peptide and proteinlevels was set to 1%; data were filtered using row based extraction. Theassay library (protein inventory) generated in this project was used forthe analysis. The HRM measurements analyzed with Spectronaut™ werenormalized using local regression normalization (Callister et al.,“Normalization Approaches for Removing Systematic Biases Associated withMass Spectrometry and Label-Free Proteomics,” J. Proteome Res. 5:277-286(2006), which is hereby incorporated by reference in its entirety).

Merging Proteomics and Transcriptomics Data

For proteomics/transcriptomics merging, gene IDs contained in thetranscriptomics data sets were matched to gene names in the mouseUniProt Swiss-Prot proteome.

Statistical Analysis for Luciferase Activity Assay and RNA ProteinCorrelation

All statistical analysis was performed with GraphPad-Prism software.Values are reported as means±SD. One-way ANOVA with Bonferronicorrection (*p<0.05 considered significant) was used for comparisonsamong groups. Parson R correlation was calculated for correlationbetween changes in mRNA expression and protein levels.

Example 1—Characterizing the Ischemic Heart Transcriptome and Proteome

To characterize the dynamics of the heart LV transcriptome and proteomepost-MI, changes in gene expression and protein levels in the LV of miceat 4-hours and at 24-hours post-MI were analyzed and compared to changesto the LV from sham-operated mice (FIG. 1A). In total, 14,552 genes and2,397 proteins were detected in the analyzed samples. When comparing thetwo datasets, 2,272 genes were found with corresponding proteins. Out ofall genes with corresponding proteins, 239 genes and 120 proteins weredifferentially expressed (q value<0.05) 4-hours post-MI. 24-hourspost-MI, 1,702 genes and 272 proteins were differentially expressed. Ahierarchical clustering dendrogram of gene expression (FIG. 1B, FIG. 2A)and protein levels (FIG. 1C) shows that in both cases the experimentalgroups cluster together, demonstrating significant differences in thetranscriptome and proteome post-MI.

Pearson correlation analysis revealed a positive correlation betweenchanges in gene expression and protein expression both 4-hours post-MI(r square=0.02, FIG. 1D) and 24-hours post-MI (r square=0.13, FIG. 1E).Significant correlation was found between the changes in gene expressionand protein levels in the ischemic heart post-MI and in searching for a5′ UTR element that may elevate modRNA translation in the heart post-MI.

Example 2—Identification of 5′ UTR Elements that Increase Translation ofmodRNA in the Heart Post-Myocardial Infarction (MI)

To identify genes with a significant non-correlation relationshipbetween mRNA and protein expression, a screen for (i) genes that encodeproteins with elevated levels 4-hours or 24-hours post-MI (foldchange>2), (ii) mRNA that is downregulated at 4-hours or 24-hourspost-MI (fold change<0.64), and (iii) a 5′ UTR shorter than 100 bp wascarried out. 3 and 18 genes, each of which displayed high proteinexpression accompanied by lower or unchanged mRNA level as compared tosham-operated hearts, were identified at 4-hours (FIG. 1F) and 24-hours(FIG. 1G) post-MI, respectively. Five genes with the shortest 5′ UTRamong those 19 genes (as Fmd5 and Serpina 1b were present in both the4-hour and 24-hour screen results) were identified (Gsn, Pzf, Serpina1b, Fn3k, and Ces1d) (FIGS. 1F-1G; Table 4). The chosen genes had anupregulated protein expression that was unrelated to their mRNAexpression, post-myocardial infarction. In addition, Ces1d expressionresults were validated using qPCR and western blot showing similar mRNAand protein expression as evaluated by RNAseq and proteomic analysis(FIGS. 2B-2D).

TABLE 4 5′ UTR Sequence of Selected Genes from FIG. 1F and FIG. 1G.SEQ ID Gene Name 5′ UTR Sequence^(†) NO. Ces1dAGGAGGCGGGTCCCCTGGTCCACAACAGAAGCATTGCTAAAGC 1 AGCAGATAGC

TTGTCCTTCCACA Gsn GCTAGGGCGGGATGGGACGGCCGGTTACTTAAAGGTTGGGGCG 54ACCAAGGGTCCGCGGCCGCAGCCTGGGTCCTCACCGTCGCC PzpCAAGGATCAGAGTTCGGGGGCTGAGGGCTCAGACGTTCTTCTC 55 TGCCCTCTCCACC Serpina 1bATATCCCCCTTGGCTCCCACTGCTTAAATACAGACTAGGAGAG 56GGCTCTGTCTCCTCAGCCTCGGTCACCACCCAGCTCTGGGACA GCAAGCTGAAA Fn3kTGCGTCACCTGACCGCATTCTGCACCTCAACTCTCC 57 ^(†)RNA Element D of thecarboxylesterase 5′ UTR sequence is shown in bold underline in the aboveTable 4.

Example 3—The 5′ UTR of Ces1d Increases In Vitro Expression of modRNA inCardiomyocytes

To evaluate the translational efficiency of the 5′ UTR of genesidentified in Table 4, five Luc modRNA constructs, each carrying the 5′UTR from one of the selected genes (i.e., Gsn, Pzf, Serpina 1b, Fn3k,and Ces1d), were designed and generated (FIG. 3A). A control Luc modRNAconstruct (Luc-Control) that carries an artificial 36 nucleotide 5′ UTRcommonly used for in vitro modRNA production was also generated (see,e.g., Warren et al., “Highly Efficient Reprogramming to Pluripotency andDirected Differentiation of Human Cells with Synthetic Modified mRNA,”Cell Stem Cell 7:618-630 (2010), which is hereby incorporated byreference in its entirety). In vitro screening showed that all LucmodRNA constructs tested allowed modRNA protein translation in neonatalrat cardiomyocytes (FIGS. 3B-3C). However, Luc modRNA constructscomprising the 5′ UTR from Gsn, Pzf, Serpina 1b, or Fn3k demonstratedsignificantly lower translational efficiency when compared to theLuc-Control modRNA construct (FIG. 3C). Interestingly, the Luc modRNAconstruct comprising the 5′ UTR of Ces1d (Luc-Ces1d) showed a 23%increase in modRNA translation, as compared to the Luc-Control modRNAconstruct (FIG. 3C, FIGS. 4A-4B).

To confirm that the increase in modRNA translation using the Luc-Ces1dconstruct was not due to differential transfection efficiencies, aparallel in vitro experiment was carried out with co-transfection ofFirefly Luc 5′ UTR modRNA constructs and a Renilla Luc modRNA constructcomprising a control 5′ UTR (as an internal control) in neonatal ratcardiomyocytes (Renilla Luc-Control) (FIG. 4A). 24 hourspost-transfection, the IVIS® system was used to measure simultaneouslythe translation of the two different luciferase (firefly and Renilla)modRNA, in vitro (FIG. 4B). Similar to the results shown in FIG. 3F, theLuc-Ces1d modRNA construct produced a significantly higher firefly Lucsignal (which is indicative of a significantly higher translationefficiency) as compared to the Luc-Control modRNA construct, without acorresponding significant change in the Renilla Luc signal producedusing the Renilla Luc-Control modRNA construct (FIGS. 4B-4C). Theseresults indicate that the high translation efficiency of the modRNAconstructs is due to the use of the Ces1d 5′ UTR and not due todifferent transfection efficiency.

To further confirm this finding, eGFP modRNA constructs comprising the5′ UTR of Ces1d (eGFP-Ces1d) or the artificial 36 nucleotide 5′ UTRdescribed above (eGFP-Control) were generated. The eGFP translationlevels of the eGFP-Ces1d and eGFP-Control constructs were evaluated bywestern blot in neonatal rat cardiomyocytes. Similar to the Luc modRNAresults, eGFP-Ces1d showed a 22% increase in modRNA translation incomparison to eGFP-Control (FIG. 3D).

Example 4— The 5′ UTR of Ces1d Increases In Vivo Expression of modRNA inCardiomyocytes

A mouse myocardial infarction model was used to further evaluate thetranslational efficiency of the three Luc modRNA constructs that showedthe highest translation levels in vitro (Luc-Pzp, Luc-Serpina 1b, andLuc-Ces1d) (FIGS. 3B-3D). The in vivo translation efficiency of Luc-Pzp,Luc-Serpina 1b, Luc-Ces1d, and Luc-Control modRNA constructs wasmeasured 24-hours, 48-hours, 72-hours, and 96-hours post-MI (FIG. 3E).FIG. 3F demonstrates that injection with Luc-Ces1d modRNA resulted insignificantly (2-fold) higher luciferase signals, which indicate asignificantly higher translation of the modRNA construct than eitherLuc-Control modRNA, Luc-Pzp modRNA, or Luc-Serpina 1b modRNA constructs.

Example 5—Pharmacokinetic Evaluation of Luc-Ces1d modRNA in Mouse Heart

To determine whether the 5′ UTR of Ces1d regulates protein translationin other contexts, Luc-Ces1d and Luc-Control modRNA-mediated translationwas evaluated in non-ischemic (FIGS. 5A-5B) and ischemic heart mousemodels (FIGS. 5C-5D, FIGS. 6A-6B). While there were no significantchanges in Luc-Ces1d modRNA translation levels in comparison withLuc-Control modRNA in the heart without MI 24 hours, 48 hours, or 72hours days post-injection (FIG. 5B), significantly more Luc-Ces1dtranslation was observed than Luc-Control translation 48 hours-dayspost-MI (FIG. 5D; FIG. 6B). These results suggest that the 5′ UTR ofCes1d may enhance modRNA translation in the heart only under ischemicconditions, like MI.

Example 6—Pharmacokinetic Evaluation of Luc-Ces1d modRNA in Mouse Liverand Kidney

The role of the 5′ UTR of Ces1d in modRNA translation under ischemicconditions in organs other than the heart was next evaluated. Sinceacute hepatic or renal ischemia may lead to hepatic or renal failure,which may be fatal, liver and kidney were chosen as representativeorgans for ischemic disease. Similar to the heart, ischemic injury inthe liver significantly increased Luc-Ces1d modRNA expression (4-hourspost-delivery) in comparison to Luc-Control modRNA expression (FIGS.6C-6D, FIGS. 7A-7D). Similar to heart, no significant differences wereseen between groups in the liver without ischemic injury (FIGS. 7A-7B).Yet, Luc-Ces1d modRNA had no significant translational differences incomparison to Luc-Control in ischemic and non-ischemic conditions in thekidney (FIGS. 6E-6F, FIGS. 7E-7H).

Example 7—RNA Element D Increases modRNA Translation Post-MyocardialInfarction

To identify the RNA element in the 5′ UTR of Ces1d responsible for thesignificantly enhanced translation of modRNA carrying the 5′ UTR ofCes1d, consensus elements conserved among different species (e.g.,mouse, rat, pig, gerbil, and human) were examined. Interestingly, fourout of the five elements identified (Elements B-E) were conserved amongspecies (FIG. 8A). Based on this information, five Luc modRNAconstructs, each carrying a different RNA element of Ces1d as its 5′UTR, were generated (i.e., Luc-Element A, Luc-Element B, Luc-Element C,Luc-Element D, and Luc-Element E modRNA constructs). The translationabilities of each of those constructs was evaluated in neonatal ratcardiomyocytes in comparison to Luc-Ces1d modRNA (FIGS. 8B-8C).Significantly reduced translation ability was observed for Luc-ElementA, Luc-Element B, Luc-Element C, and Luc-Element E modRNA constructs,but not for the Luc-Element D modRNA construct (FIG. 8C). It was thushypothesized that element D is the RNA element responsible for thehigher translation ability of Luc-Ces1d.

To confirm that Element D is the RNA element responsible for the highertranslation ability of Luc-Ces1d modRNA, the expression of Luc-Ces1d andLuc-Element D was compared to the expression of Luc-Control in anischemic heart model over three days (FIG. 8D). FIG. 8E demonstratesthat Luc modRNA constructs comprising the 5′ UTR of Ces1d or Element Dof Ces1d have increased translation compared to Luc-Control modRNA.Surprisingly, Element D of Ces1d alone has a significantly highertranslation rate in the heart at 3 days post-MI (FIG. 8E). Overall,combining the three day readouts, Luc-Element D translation in the heartpost-MI was 2.5-fold higher than the widely used artificial 5′ UTR,Luc-Control. However, Luc-Element D failed to increase translation morethan Luc-Ces1d in liver ischemic injury (FIGS. 9A-9B) and over Ces1d-Lucor Luc-Control in heart non-ischemic injury (FIGS. 10A-10B).

Discussion of Examples 1-7

The use of modRNA as a gene delivery tool is growing in the field oftherapeutic medicine. The results presented herein suggest that modRNAmay be employed to induce cardiac protection, cardiovascularregeneration, and cardiovascular regeneration post-ischemic injury(Hadas et al., “Modified mRNA as a Therapeutic Tool to Induce CardiacRegeneration in Ischemic Heart Disease,” Wiley Interdiscip. Rev. Syst.Biol. Med. 9 (2017), which is hereby incorporated by reference in itsentirety). The immediate delivery of VEGF-A modRNA in a MI mouse modelhas been shown to lead to the induction of cardiovascular regeneration(Zangi et al., “Modified mRNA Directs the Fate of Heart Progenitor Cellsand Induces Vascular Regeneration after Myocardial Infarction,” Nat.Biotechnol. 31:898-907 (2013), which is hereby incorporated by referencein its entirety). A follow-up large animal study showed a significantimprovement of cardiac function when VEGF-A modRNA was delivered oneweek post-MI (Carlsson et al., “Biocompatible, Purified VEGF-A mRNAImproves Cardiac Function after Intracardiac Injection 1 WeekPost-myocardial Infarction in Swine,” Mol. Ther. Methods Clin. Dev.9:330-346 (2018), which is hereby incorporated by reference in itsentirety). Recently, intradermal VEGF-A modRNA delivery in patientssuffering from type 2 diabetes was shown to be safe and to possiblypromote angiogenesis (Gan et al., “Intradermal Delivery of Modified mRNAEncoding VEGF-A in Patients with Type 2 Diabetes,” Nat. Commun. 10:871(2019), which is hereby incorporated by reference in its entirety).

VEGF-A modRNA is now being evaluated in Phase 2a human clinical trialsto improve cardiac function in patients with heart failure. In parallel,other groups are using modRNA to deliver different target genes inpreclinical studies of different liver diseases, including models forfactor IX deficiency hemophilia B (DeRosa et al., “Therapeutic Efficacyin a Hemophilia B Model Using a Biosynthetic mRNA Liver Depot System,”Gene Ther. 23:699-707 (2016), which is hereby incorporated by referencein its entirety); acute intermittent porphyria (Jiang et al., “SystemicMessenger RNA as an Etiological Treatment for Acute IntermittentPorphyria,” Nat. Med. 24:1899-1909 (2018), which is hereby incorporatedby reference in its entirety); glycogen storage disease type 1A (Rosemanet al., “G6PC mRNA Therapy Positively Regulates Fasting Blood Glucoseand Decreases Liver Abnormalities in a Mouse Model of Glycogen StorageDisease 1a,” Mol. Ther. 26:814-821 (2018), which is hereby incorporatedby reference in its entirety); thrombotic thrombocytopenic purpura(Liu-Chen et al., “mRNA Treatment Produces Sustained Expression ofEnzymatically Active Human ADAMTS13 in Mice,” Sci. Rep. 8:7859 (2018),which is hereby incorporated by reference in its entirety); alpha-1antitrypsin deficiency (Connolly et al., “SERPINA1 mRNA as a Treatmentfor Alpha-1 Antitrypsin Deficiency,” J. Nucleic Acids2018:8247935(2018), which is hereby incorporated by reference in itsentirety); Crigler-Najjar syndrome type 1 (Apgar et al., “QuantitativeSystems Pharmacology Model of hUGT1A1-modRNA Encoding for the UGT1A1Enzyme to Treat Crigler-Najjar Syndrome Type 1,” CPT PharmacometricsSyst. Pharmacol. 7:404-412 (2018), which is hereby incorporated byreference in its entirety); and urea cycle disorders (Prieve et al.,“Targeted mRNA Therapy for Ornithine Transcarbamylase Deficiency,” Mol.Ther. 26:801-813 (2018), which is hereby incorporated by reference inits entirety) with target genes of FIX, PBGD, glucose-6-phosphatase,ADAMTS13, SERPINA1, bilirubin-UGT, ornithine, and transcarbamoylase,respectively.

As described above, both heart and liver diseases have been heavilystudied in the search for new treatments using modRNA. One obstacle inmoving to large animal and clinical trials that is related to modRNA isthe need for large amounts of modRNA to transfect human or large animalhearts and livers. In addition, due to the short expression of modRNA,it might need to be administered several times. To reduce the need toadminister large amounts and/or multiple doses of therapeutic modRNA, itis desirable to improve modRNA translation such that therapeuticallyeffective amounts of protein may be effectively translated fromrelatively low amounts of modRNA.

Several elements within RNA are responsible for regulating translationincluding, e.g., nucleotide type, poly A tail length, 5′ UTR, 3′ UTR,and cap analog structures. The results presented herein demonstrate thatreplacing pseudouridine with 1-M-pseudouridine in modRNA results inhigher modRNA translation in the heart when 1-M-pseudouridine is beingused (Sultana et al., “Optimizing Cardiac Delivery of Modified mRNA,”Mol. Ther. 25(6):1306-1315 (2017), which is hereby incorporated byreference in its entirety). In addition, it was found that a longerpoly-A tail can increase modRNA translation in vivo. Examples 1-7 hereindescribe a novel screening method that compares proteomic andtranscriptomic analysis to identify 5′ UTR sequences that can increasemodRNA translation in ischemia more effectively than the widely usedartificial 5′ UTR that has been used in previous modRNA publications(Sultana et al., “Optimizing Cardiac Delivery of Modified mRNA,” Mol.Ther. 25(6):1306-1315 (2017); Hadas et al., “Optimizing Modified mRNA InVitro Synthesis Protocol for Heart Gene Therapy,” Mol. Ther. MethodsClin. Dev. 14(13):300-305 (2019); Zangi et al., “Modified mRNA Directsthe Fate of Heart Progenitor Cells and Induces Vascular Regenerationafter Myocardial Infarction,” Nat. Biotechnol. 31:898-907 (2013);Kormann et al., “Expression of Therapeutic Proteins after Delivery ofChemically Modified mRNA in Mice,” Nat. Biotechnol. 29:154-157 (2011);Kormann et al., “Expression of Therapeutic Proteins After Delivery ofChemically Modified mRNA in Mice,” Nat. Biotechnol. 29:154-157 (2011);Carlsson et al., “Biocompatible, Purified VEGF-A mRNA Improves CardiacFunction after Intracardiac Injection 1 Week Post-myocardial Infarctionin Swine,” Mol. Ther. Methods Clin. Dev. 9: 330-346 (2018); and Gan etal., “Intradermal Delivery of Modified mRNA Encoding VEGF-A in Patientswith Type 2 Diabetes,” Nat. Commun. 10:871 (2019), which are herebyincorporated by reference in their entirety).

The results presented herein show a positive correlation at both 4-hoursand 24-hours post-MI in mRNA levels and protein intensity in theischemic heart. 19 negatively correlating genes, in which mRNA levelswere either reduced or unchanged while their protein levels wereupregulated at 4-hours and 24-hours post-MI, were also identified. Theaverage 5′ UTR length is ˜100 to ˜220 nucleotides across species (Pesoleet al., “Structural and Functional Features of Eukaryotic mRNAUntranslated Regions,” Gene 276:73-81 (2001), which is herebyincorporated by reference in its entirety). In vertebrates, longer 5′UTRs tend to be associated with poor translation (Davuluri et al., “CARTClassification of Human 5′ UTR sequences,” Genome Res. 10:1807-1816(2000), which is hereby incorporated by reference in its entirety).Therefore, five genes with the shortest 5′ UTRs were selected forevaluation (<100 base pairs) (FIGS. 1A-1G).

Negative correlation between mRNA levels and protein expression has beenreported, especially when internal or external stimuli triggeralteration in translation of specific genes. VEGF-A has been shown to bea stress-induced protein in many conditions such as hypoxia andhypoglycemia (Shweiki et al., “Induction of Vascular Endothelial GrowthFactor Expression by Hypoxia and by Glucose Deficiency in MulticellSpheroids: Implications for Tumor Angiogenesis,” Proc. Natl. Acad. Sci.USA 92:768-772 (1995) and Akiri et al., Regulation of VascularEndothelial Growth Factor (VEGF) Expression is Mediated by InternalInitiation of Translation and Alternative Initiation of Transcription,”Oncogene 17:227-236 (1998), which are hereby incorporated by referencein their entirety). Other genes that undergo translation changes inresponse to internal or external signals are PDGF2 and TGFβ (Tobin etal., “Consequences of Altered TGF-beta Expression and Responsiveness inBreast Cancer: Evidence for Autocrine and Paracrine Effects,” Oncogene21:108-118 (2002), which is hereby incorporated by reference in itsentirety). During the embryonic stage, when most organ development andcell differentiation takes place, translation regulation plays a keyrole by altering the expression levels of specific mRNA subsets during acertain time frame whereas the bulk of transcripts remain unaffected(Jackson et al., “The Mechanism of Eukaryotic Translation Initiation andPrinciples of Its Regulation,” Nat. Rev. Mol. Cell Biol. 11:113-127(2010); Sonenberg & Hinnebusch, “Regulation of Translation Initiation inEukaryotes: Mechanisms and Biological Targets,” Cell 136:731-745 (2009);and Gebauer & Hentze, “Molecular Mechanisms of Translational Control,”Nat. Rev. Mol. Cell Biol. 5:827-835 (2004), which are herebyincorporated by reference in their entirety).

The results presented herein identify several 5′ UTRs that may allowtranslation of modRNA in the heart or liver post ischemic injury. BothPzp and Serpina 1b 5′ UTR showed non-significant and similar in vivotranslation ability as a control 5′ UTR sequence (FIGS. 3E-3F). Sincethe control 5′ UTR used in the studies described herein is the mostcommonly used 5′ UTR in the modified RNA field (Zangi et al., “ModifiedmRNA Directs the Fate of Heart Progenitor Cells and Induces VascularRegeneration after Myocardial Infarction,” Nat. Biotechnol. 31:898-907(2013); Carlsson et al., “Biocompatible, Purified VEGF-A mRNA ImprovesCardiac Function after Intracardiac Injection 1 Week Post-myocardialInfarction in Swine,” Mol. Ther. Methods Clin. Dev. 9: 330-346 (2018);Kondrat et al., “Synthesis of Modified mRNA for Myocardial Delivery,”Methods in Molecular Biology 1521:127-138 (2017); Lui et al., “DrivingVascular Endothelial Cell Fate of Human Multipotent Isl1+HeartProgenitors with VEGF Modified mRNA,” Cell Res. 23:1172-1186 (2013);Magadum et al., “Ablation of a Single N-Glycosylation Site in Human FSTL1 Induces Cardiomyocyte Proliferation and Cardiac Regeneration,” Mol.Ther. Nucleic Acids 13:133-143 (2018); Mohamed et al., “Regulation ofCell Cycle to Stimulate Adult Cardiomyocyte Proliferation and CardiacRegeneration,” Cell 173:104-116 e112 (2018); and Turnbull et al.,“Myocardial Delivery of Lipidoid Nanoparticle Carrying modRNA InducesRapid and Transient Expression,” Mol. Ther. 24:66-75 (2016), which arehereby incorporated by reference in their entirety), the resultspresented herein indicate how well the selected 5′ UTR sequencesperformed relative to a premium control.

The studies presented herein focused on the 5′ UTR of Ces1d as anenhancer of modRNA translation in cardiac and hepatic ischemicconditions (FIGS. 3A-3F; FIGS. 5A-5D; and FIGS. 7A-7H). Ces1d belongs toa family of carboxylesterases, which have important roles in lipidmetabolism and hydrolyze endogenous esters and thioesters.Carboxylesterases are known for their involvement in environmentaldetoxification as well as pro-drug metabolism. Ces1d is the functionalmouse ortholog of human CESJ and has similar protein expression profilesin different cells and tissues. Several of Ces1d's roles are directlyassociated with lipid metabolism (Dominguez et al., “IntegratedPhenotypic and Activity-Based Profiling Links Ces3 to Obesity andDiabetes,” Nat. Chem. Biol. 10:113-121 (2014); Lian et al., “Ces1dDeficiency Protects Against High-Sucrose Diet-Induced HepaticTriacylglycerol Accumulation,” J. Lipid Res. 60:880-891 (2019); andMarrades et al., “A Dysregulation in CES1, APOE and Other LipidMetabolism-Related Genes is Associated to Cardiovascular Risk FactorsLinked to Obesity,” Obes. Facts 3:312-318 (2010), which are herebyincorporated by reference in their entirety), which is an importantprocess for normal heart function. Fatty acids are the preferredsubstrates under aerobic conditions (Ford, “Alterations in MyocardialLipid Metabolism During Myocardial Ischemia and Reperfusion,” Prog.Lipid Res. 41:6-26 (2002), which is hereby incorporated by reference inits entirety). As Ces1d takes part in lipid metabolism, which MI alters,it is hypothesized that Ces1d mRNA is triggered toward improvedtranslation by ischemia in the heart post-MI. The results presentedherein show that Element D is the RNA element in Ces1d that isresponsible for elevating mRNA translation post-MI (FIGS. 7A-7H). It istherefore desirable to evaluate Element D and the 5′ UTR of Ces1d in thecontext of different ischemic conditions, as well as in organs otherthan the heart, liver, and kidney. The fact that the 5′ UTR of Ces1draised translation in the ischemic heart and liver, but not the kidney,is interesting, as all three organs primarily uses fatty acid oxidationfor energy. This result may indicate that each organ and physiologicalcondition will need a separate evaluation using approaches similar tothose described herein.

The results presented herein identify the 5′ UTR of Ces1d, and element Din the 5′ UTR of Ces1d as RNA elements that improve modRNA translationin the heart and liver post-ischemic injury. This may have clinicalapplications, as both organs are heavily targeted with modRNA indifferent cardiac and hepatic diseases. These results inform the designof superior modRNA constructs which may carry the 5′ UTR of Ces1d orElement D of Ces1d for preclinical studies in ischemic cardiac and liverdiseases. The results presented herein also provide a platformtechnology for screening for superior 5′ UTRs in different organs undernormal or abnormal physiological conditions or diseases.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A nucleic acid molecule comprising: a first nucleic acid sequencecomprising at least a portion of a 5′ untranslated region (5′ UTR) of acarboxylesterase gene, wherein the carboxylase is a carboxylase 1D(Ces1D) or a carboxylesterase 1 (CES1), and a second nucleic acidsequence encoding a protein of interest, wherein the second nucleic acidsequence is heterologous to and operatively coupled to the first nucleicacid sequence.
 2. The nucleic acid molecule according to claim 1,wherein said first and second nucleic acids are modified mRNAs(modRNAs).
 3. The nucleic acid molecule according to claim 2, whereinthe modRNAs comprise pseudouridine or methylpseudouridine.
 4. Thenucleic acid molecule according to claim 1, wherein the carboxylesterasegene is a carboxylesterase 1D (Ces1d) gene.
 5. (canceled)
 6. The nucleicacid molecule according to claim 4, wherein the first nucleic acidsequence comprises the nucleic acid sequence of SEQ ID NO:1.
 7. Thenucleic acid molecule according to claim 4, wherein the first nucleicacid sequence comprises the nucleic acid sequence of SEQ ID NO:10. 8.The nucleic acid molecule according to claim 1, wherein thecarboxylesterase gene is a carboxylesterase 1 (CES1) gene.
 9. (canceled)10. The nucleic acid molecule according to claim 8, wherein the firstnucleic acid sequence comprises the nucleic acid sequence of SEQ IDNO:5.
 11. The nucleic acid molecule according to claim 8, wherein thefirst nucleic acid sequence comprises the nucleic acid sequence of SEQID NO:29.
 12. The nucleic acid molecule according to claim 1, whereinthe protein of interest is a cell cycle inducer.
 13. The nucleic acidmolecule according to claim 12, wherein the cell cycle inducer isselected from the group consisting of Lin28, Pyruvate Kinase MuscleIsozyme M2 (Pkm2), β-catenin, caERBB2, Yes Associated Protein 1 (YAP),Cyclin D1, and c-Myc.
 14. (canceled)
 15. (canceled)
 16. (canceled) 17.(canceled)
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. A method ofexpressing a protein of interest in a target cell, said methodcomprising: providing the nucleic acid molecule according to claim 1 andcontacting a target cell with the nucleic acid molecule, wherein saidnucleic acid molecule is translated to express the protein of interestin the target cell.
 22. (canceled)
 23. (canceled)
 24. The methodaccording to claim 21, wherein the target cell is an ischemic cell. 25.The method according to claim 21, wherein the target cell is acardiomyocyte or hepatocyte.
 26. (canceled)
 27. (canceled) 28.(canceled)
 29. The method according to claim 21, wherein said contactingis carried out after an ischemic event in the target cell. 30.(canceled)
 31. (canceled)
 32. (canceled)
 33. A method of treating asubject for cardiac ischemia or hepatic ischemia, said methodcomprising: providing the nucleic acid molecule according to claim 1 andcontacting the subject with the nucleic acid molecule or pharmaceuticalcomposition, wherein said nucleic acid molecule is translated to expressa protein of interest in the subject's heart or liver to treat thesubject for cardiac ischemia or hepatic ischemia.
 34. The methodaccording to claim 33, wherein said method is carried out to treat thesubject for cardiac ischemia.
 35. (canceled)
 36. The method according toclaim 33, wherein said method is carried out to treat the subject forhepatic ischemia.
 37. (canceled)
 38. (canceled)
 39. (canceled) 40.(canceled)
 41. (canceled)
 42. (canceled)
 43. The method according toclaim 33, wherein the protein of interest is a cell cycle inducer. 44.The method according to claim 43, wherein the cell cycle inducer isselected from the group consisting of Lin28 and Pyruvate Kinase MuscleIsozyme M2 (Pkm2).
 45. (canceled)
 46. (canceled)
 47. (canceled) 48.(canceled)
 49. (canceled)
 50. (canceled)
 51. (canceled)
 52. (canceled)53. (canceled)
 54. (canceled)
 55. (canceled)